Nucleotide sequences encoding mammalian calcium activated chloride channel-adhesion molecules

ABSTRACT

Nucleotide sequences which encode a mammalian lung endothelial cell adhesion molecule are disclosed. Also disclosed are nucleotide sequences which encode a lung endothelial cell adhesion molecule-associated protein. Recombinant lung endothelial cell adhesion molecule or recombinant lung endothelial cell adhesion molecule-associated protein may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant lung endothelial cell adhesion molecule-associated protein or recombinant lung endothelial cell adhesion molecule-associated protein from the culture medium.

This application is a Divisional of U.S. patent application Ser. No.09/193,562 filed on Nov. 17, 1998, now U.S. Pat. 6,309,857 which claimsthe priority of U.S. Provisional Application Ser. No. 60/065,922 filedon Nov. 17, 1997, the disclosures of which are incorporated herein byreference.

This invention was made with government support under grants CA 47G68and 09682 from the National Cancer Institute. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to nucleotide sequences encoding a familyof mammalian calcium activated chloride channels which mayalternatively, or additionally function as adhesion molecules. Moreparticularly, the invention is directed to genes isolated from bovineendothelial cells, human At endothelial cells and murine endothelialcells, which encode calcium activated chloride channel molecules andinclude the lung-endothelial cell adhesion molecules (Lu-ECAM-1) andassociated proteins.

BACKGROUND OF THE INVENTION

Calcium Activated Chloride Channels

Ion channels are not only required for normal cellular functions butalso play a critical role in numerous diseased states. For example,cystic fibrosis results when ion transport in epithelial cells ofindividuals is altered due to a genetic defect of the cystic fibrosistransmembrane conductance regulator CFTR; Knowles et al., 1983, J. Clin.Invest.71:1410-1417). Although serious airway pathology is usually theprimary cause of mortality in young adults with CF, intestinalepithelial alterations have also been observed. However, the severity oftissue lesions does not correlate with the expression of CFTR in humansor mice, suggesting the involvement of cell-specific channels inaddition to CFTR. Further support for the involvement of other channelprotein molecules in CF comes from observations that calcium activatedchloride secretion is preserved in respiratory epithelia of CF patientscompared to unaffected individuals, but is significantly reduced orabsent from CFTR-defective epithelia. These results strongly suggestthat an alternative non-CFTR regulated chloride channel activity mightaccount for attenuating CF disease in some tissues. Thus, a need existsfor identification, isolation and functional analysis of alternativechloride channels.

Adhesion Molecules

It is apparent that endothelial cell adhesion molecules may havefunctions in addition to their adhesive functions. For example,integrins have transmembrane signaling capacities which may play a rolein the adherence process. However, the primary function of endothelialcell adhesion molecules is adherence to a substrate such as (a) topromote adherence of endothelial cells to basement membrane, (b) topromote vascular arrest and to facilitate extravasation of leukocytessuch as during an immune response, and (c) to promote homing oflymphocytes to a particular lymphoid tissue. Other molecules may play arole in controlling adherence of endothelial cells. For example,chloride ion channels are thought to be involved in a signaling cascadewhen lymphatic endothelial cells begin to adhere to a substrate (Martinet al., 1996, Microvasc. Res. 52:200-9).

There is considerable evidence that metastatic nonlymphoid tumor cellsmimic leukocytes in recognizing and adhering to one or more endothelialcell adhesion molecules to migrate in blood vessels, to arrest invascular areas of organs which may provide the microenvironmentconducive for metastatic growth, and to extravasate into surroundingtissues. An example of such an endothelial cell adhesion molecule whichpromotes adhesion of tumor cells and mediates metastasis islung-endothelial cell adhesion molecule (Lu-ECAIVI-1). Lu-ECAM-1 is a 90kilodalton (kDa) integral membrane protein constitutively expressedprimarily in endothelial cells of pleural and subpleural microvessels.Both in vitro studies and in vivo studies indicate thatLu-ECAM-1-expressing endothelial cells promote adhesion of certainlung-colonizing tumor cells in a manner that is consistent with theexpression level of the adhesion molecule and the metastatic propensityof tumor cells. For example, in an in vitro tumor cell/endothelial celladhesion assay, highly lung metastatic B1G-F10 melanoma cells bind tolung-matrix-modulated endothelial cells expressing Lu-ECAM-1 insignificantly larger numbers than their intermediate or lowlung-metastatic counterparts (B1G-L8-F1O and B1GFO, respectively; Zhu etal., 1991, Proc. Nati. Acad. Sd. USA 88:9568-720). Such binding appearsto be calcium (Ca²⁺) dependent. Further, anti-Lu-ECAM-1 monoclonalantibodies significantly inhibit adhesion of B1GF10 melanoma cells toLu-ECAM-1 expressing endothelial cells in culture (Zhu et al., 1991,supra) Anti-Lu-ECAM-1 monoclonal antibodies are also efficient inpreventing metastatic colonization of the lungs by highlylung-metastatic B1GF10 cells in a standard animal model for metastasis(Zhu et al., 1991, supra). Lu-ECAM-1, affinity purified from detergentextracts of bovine aortic endothelial cells, was used to immunize mice.The immunized mice showed an inhibition of metastatic colonization ofthe lungs by B1GF10 melanoma cells, the efficiency of which wasdependent upon the anti-Lu-ECAN-l serum titer (Zhu et al., 1992, J.Clin. Invest. 89:1718-1724). Lu-ECAM-1 appears to be the endothelialcell adhesion molecule for metastatic tumor cells that express theligand/34 integrin subunit (and possibly other ligands) including, butnot limited to, lung-metastatic breast tumor cells, and lung-metastaticmelanoma tumor cells.

Anti-adhesion therapy may be used to interfere with adhesion betweenorgan-specific endothelial cells and blood-borne cancer cells inpreventing the formation of metastatic colony formation in organs thatsupport metastatic cell growth. The amount of endothelial cell adhesionmolecule that can be made from detergent extracts, as well as the rateof production of the endothelial cell adhesion molecule, is generallyinsufficient for cost-effective commercial production. More efficientproduction of proteins, with a concomitant reduction in production cost,can often be achieved by producing a protein through recombinant means.In that regard, in some cases a host cell may be genetically engineeredsuch that an increased amount of the protein is produced and/or theprotein is produced in a manner which facilitates its isolation (ascompared to harvesting the protein from cell membranes).

SUMMARY OF THE INVENTION

It is an object of the invention to provide nucleotide sequences,isolated from mammalian endothelial cells, which encode molecules thatfunctions as a calcium activated chloride channel-adhesion molecule(CACC-AM).

It is also an object of the present invention to provide nucleotidesequences which are variants (including portions) of the gene comprisingthe CACC-AM, and which encode a polypeptide having substantially thebiological activity as compared to the biological activity of theCACC-AM.

It is an object of the present invention to provide a means forrecombinantly producing CACC-AM molecule.

It is an object of the present invention to provide a means forrecombinantly producing proteins associated with CACC-AM molecule.

It is a further object of the present invention to provide expressionvectors containing a nucleotide sequence that encodes a CACC-AMmolecule; or containing a nucleotide sequence which is a variant of thegene for CACC-AM, and that encodes a polypeptide having substantialbiological activity of a CACC-AM; or containing a nucleotide sequencethat encodes a protein associated with a CACC-AM.

It is an additional object of the present invention to providerecombinant host cells which contain multiple copies of a nucleotidesequence that encodes a CACC-AM molecule, wherein the CACC-AM moleculeis recombinantly produced by culturing the recombinant host cells undersuitable conditions.

Other objects, features, and advantages of the present invention willbecome apparent from the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a method for identifying clones usingpolymerase chain reaction. Also shown are restriction enzyme sites EcoRI(“R”); NdeI (“N”), PstI (“P”), and BglII (“B”).

FIG. 2A is a representation of immunoblots of bovine aortic endothelialcell proteins using either monoclonal antibody D3 (“D3”), polyclonalantibody CU11 (“11”), polyclonal antibody CU19 (“19”), polyclonalantibody R4 (“R4”), and polyclonal antibody R41 (“R41”).

FIG. 2B is a representation of Lu-ECAM-1 untreated (“−”)or Lu-ECAM-1treated with N-glycosidase F (“+”) followed by immunoblot analysis usingpolyclonal antibody R4; and Lu-ECAM-1-associated proteins untreated(“−”) or Lu-ECAM-1-associated proteins treated with N-glycosidase F(“+”) followed by immunoblot analysis using polyclonal antibody R41.

FIG. 3A is a representation of bovine aortic endothelial cells eitheruntreated (“−”) or treated with a crosslinker (“+”) followed byimmunoblot analysis using either polyclonal antibody R4 (“R4”), orpolyclonal antibody R41 (“R41”).

FIG. 3B is a representation of bovine aortic endothelial cells whichwere surface-biotinylated in the absence of (“−”) or presence of (“+”) acrosslinker followed by detection with streptavidin-horseradishperoxidase.

FIG. 4A is a representation of a ethidium bromide stained agarose gelcontaining the results of reverse transcriptase polymerase chainreaction analysis of bovine aortic endothelial cells (“BAEC”), lungtissue, tracheal epithelium, and spleen tissue using Lu-ECAM-1 specificprimer pairs L1, and L2.

FIG. 4B is a representation of a ethidium bromide stained agarose gelcontaining the results of reverse transcriptase polymerase chainreaction analysis of bovine aortic endothelial cells (“BAEC”), lungtissue, tracheal epithelium, and spleen tissue using bovine trachealchloride channel (“Ca-CC”) specific primer pairs T1, and T2.

FIG. 5 is a bar graph illustrating lung-metastatic tumor cell adhesionto wild type Lu-ECAM-1 in the presence or absence of anti-Lu-ECAM-1 mAb6D3; and lung-metastatic tumor cell adhesion to recombinant Lu-ECAM-1 inthe presence or absence of anti-LuECAM-1 mAb 6D3.

FIG. 6 is a representation of the expression of mCLCA1 by in vitrotranslation (A) and in transfected HEK293 cells (B)

FIG. 7 is a representation of the biochemical analysis of hCLCA1 proteinfor in vitro translated (a), c-myc tagged hCLCA1 transfected HEK293 (b),and surface expression of c-myc tagged hCLCA1.

FIG. 8 is a representation of biochemical analysis of the hCLCA2 proteinfor in vitro translation (a) and immunoblot detection of myc taggedhCLCA2 constructs in HEK293 cells (b)

FIG. 9 is a representation of whole cell currents in mCLCA1 transfectedHEK293 cells.

FIG. 10 is an illustration of the summary the effects of inhibitors onmCLCAl current expression.

FIG. 11 is a representation of whole cell currents in hCLCA1-transfectedHEK293 cells.

FIG. 12 is an illustration of the summary the effects of inhibitors onhCLCA1 current expression.

FIG. 13 is an illustration of the electrohpysiological analysis ofhCLCA2.

FIGS. 14A and B is a representation of a comparison of the amino acidsequences of the calcium activated chloride channels, hCLCA1 (SEQ IDNO:27); hCLCA2 (SEQ ID NO:31); bCLCA1 (SEQ ID NO:46); Lu-ECAM-1 (SEQ IDNO:1); and mCLCA1 (SEQ ID NO:33).

DETAILED DESCRIPTION

Definitions “Precursor” is a term used in conjunction with“lung-endothelial cell adhesion molecule” hereinafter for the purposesof the specification and claims to refer to a sequence of amino acidsbound to and located upstream from the N-terminal portion of the matureform of a lung-endothelial cell adhesion molecule, wherein the removalof this sequence results in the formation of the “mature form” of thelung-endothelial cell adhesion molecule. A precursor protein is a formof a lung-endothelial cell containing a prepro-region. The prepro-regionis made up of amino acids comprising a signal sequence, wherein thesignal sequence is cleaved to form the mature form of a lung-endothelialcell adhesion molecule.

“Calcium activated chloride channel—adhesion molecule” or “CACC-AM” is aterm used hereinafter for the purposes of the specification and claimsto mean a molecule isolated from mammalian endothelial cells that whenexpressed in cells induces the expression of calcium activated chlorideconductance channels.

“Calcium activated chloride channel (s)” is a term used for the purposesof the specification and claims to mean chloride channels whoseconductance is activated by calcium as judged by inhibition ofconductance by DIDS, DTT or niflumic acid.

“Recombinant calcium activated chloride channel-adhesion molecule” or“Recombinant CACC-AM” is a term used hereinafter for the purposes of thespecification and claims to refer to a CACC-AM molecule produced from aheterologous cell (e.g., other than from vascular endothelial cells),wherein the heterologous cell has been genetically engineered to containa nucleotide sequence that encodes a CACC-AM molecule.

“Recombinant calcium activated chloride channel-adhesionmolecule-associated protein” or “recombinant CACC-AM-associatedmolecule” is a term used hereinafter for the purposes of thespecification and claims to refer to a CACC-AM associated proteinproduced from a heterologous cell (e.g., other than from vascularendothelial cells) wherein the heterologous cell has been geneticallyengineered to contain a nucleotide sequence that encodes a CACC-AMassociated molecule. “Lung-endothelial cell adhesion molecule-associatedprotein” is a term used hereinafter for the purposes of thespecification and claims to refer to a protein which (a) is smaller inkilodaltons than the mature form of the lung-endothelial cell adhesionmolecule, as determined by, for example, sodium dodecyl polyacrylamidegel electrophoresis (SDS-PAGE) or amino acid analysis; (b) is encoded bymessages that also encode the lung-endothelial cell adhesion molecule;(c) is antigenically distinct from the lung-endothelial cell adhesionmolecule; and (d) is extracellularly associated in a complex (e.g,specific binding) with the lung-endothelial cell adhesion molecule.

By the term “operably linked” is meant, for the purposes of thespecification and claims to refer to the chemical fusion (restrictionwith subsequent ligation) or synthesis of heterologous DNA with anucleotide sequence that encodes a lung-endothelial cell adhesionmolecule or a lung-endothelial cell adhesion molecule-associated proteinsuch that the resultant recombinant DNA molecule is formed in a properorientation and reading frame for the nucleotide sequence to betranscribed into functional RNA. In the construction of the recombinantDNA molecule, it is generally preferred to position a promoter at adistance upstream from the initial codon of the nucleotide sequence thatis approximately the same as the distance in its natural setting (e.g.,in an endothelial cell). However, as known in the art, some variation inthe distance can be accommodated without loss of promoter function.Likewise, it is generally preferred to position an enhancer element at adistance upstream from the promoter, or incorporated into the promotersequences as a promoter element, or located between the promoter and theDNA molecule to be expressed. However, as known in the art, somevariation in the placement can be accommodated without loss of theenhancer element's function. “Expression control sequences” is meant,for the purposes of the specification and claims to refer to a promoteror promoter-enhancer combination.

By the term “expression vector” is meant, for the purposes of thespecification and claims to refer to a DNA molecule which is operablylinked to a nucleotide sequence that encodes one or more recombinantproteins comprising a lung-endothelial cell adhesion molecule and/or alung-endothelial cell adhesion molecule-associated protein such that theproduction of the recombinant protein is effected in a suitable host.The vector may include, but is not limited to, a plasmid, phage, or apotential genomic insert.

By the terms “degeneracy substitutions”, for the purposes of thespecification and claims to refer to the base pair changes(substitutions) in the nucleotide sequence such as a change in one ormore bases of a triplet codon (e.g., third base degeneracy) resulting inthe encoding of the same amino acid as before the change, or a changeresulting in the encoding of a conservative substitution in the aminoacid sequence encoded. With respect to such variations, and asappreciated by those skilled in the art, because of third basedegeneracy, almost every amino acid can be represented by more than onetriplet codon in a coding nucleotide sequence. Thus, in nature or bymutagenic means, the nucleotide sequence be modified slightly insequence (e.g., substitution of a nucleotide in a triplet codon), andyet still encode its respective gene product of the same amino acidsequence as encoded by the disclosed nucleotide sequences.

Further, the nucleotide sequence may have minor base pair changes whichmay result in variation (conservative substitution) in the amino acidsequence encoded. Such conservative substitutions are not expected tosubstantially alter the biological activity of the gene product. A“conservative substitution” for the purpose of the specification andclaims means modification of one or more amino acids are such that thetertiary configuration of the recombinant protein is substantiallyunchanged. Conservative substitutions is defined by aforementionedfunction, and includes substitutions of amino acids having substantiallythe same charge, size, hydrophilicity, and/or aromaticity as the aminoacid replaced. Such substitutions, known to those of ordinary skill inthe art, include glycine-alanine-valine; isoleucine-leucine;tryptophan-tyrosine; aspartic acid-glutamic acid; arginine-lysine;asparagine-glutamine; and serine-threonine. It is noted that anucleotide sequence according to the present invention encodes amammalian Lu-ECAM-1 as to be described more fully herein, and does notencompass the nucleotide sequence encoding the bovine trachealepithelial chloride channel described recently (Cunningham et al., 1995,J. Biol. Chem. 270:31016-26).

By the terms “% identity of amino acid sequence” are meant, for thepurposes of the specification and claims to refer to the percent ofamino acid positions that are identical between two amino acid sequencesas determined by sequence comparisons performed using algorithms knownto those skilled in the art.

By the terms “% identity of nucleotide sequence” are meant, for thepurposes of the specification and claims to refer to the percent ofnucleotide base pair positions that are identical between two nucleotidesequences as determined by sequence comparisons performed usingalgorithms known to those skilled in the art.

By the term “substantially” is used in conjunction with the biologicalactivity (e.g., adhesive function or chloride ion channel function) tomean, for the purposes of the specification and claims, to refer toretaining a degree of the biological activity ranging from approximately50% of the activity to greater than 100% of the activity, in relation tothe molecule with which it is compared.

By the term “unexpectedly improved” is used in conjunction with thebiological activity (e.g., adhesive function or chloride ion channelfunction) of a recombinant protein to mean, for the purposes of thespecification and claims, to refer to a degree of the biologicalactivity which is approximately greater or equal to 30% more biologicalactivity than that of the molecule to which it is compared, and whichimprovement in activity was unforeseen for this recombinant protein.

The present invention relates to nucleotide sequences and variantsthereof that encode a polypeptide which is a calcium activated chloridechannel and/or has adhesion properties. In accordance with thisinvention, nucleotide sequences encoding Lu-ECAM-1/mouse calciumactivated chloride channel (mCLCA), and human calcium activated chloridechannel molecules (hCLCA1, hCLCA2, and hCLCA3) are disclosed. Thenucleotide sequences have been derived from bovine aortic endothelialcells, from murine aortic endothelial cells, or from human endothelialcells. In one embodiment, a nucleotide sequence of the presentinvention, SEQ ID NO:1, contains sequences that encode either Lu-ECAM-1or Lu-ECAM-1-associated protein. From SEQ ID NO:1, the lung-endothelialcell adhesion molecule precursor is deduced to be approximately 905amino acids (SEQ ID NO:2). Cleavage of the signal peptide (amino acids−21 to −1 of SEQ ID NO:2) from the lung-endothelial cell adhesionmolecule precursor, and subsequent post-translational processing,results in a Lu-ECAM-1 of about 799 amino acids (amino acid 1 to aminoacid 799 of SEQ ID NO:2) and with a predicted molecular size ofapproximately 87 kDa. It was also discovered during the development ofthe invention that a SEQ ID NO:1 encodes a Lu-ECAM-1-associated protein(SEQ ID NO:3) which, depending on the glycosylation pattern, has anapparent molecular size (e.g., as determined by SDS-PAGE) ranging fromabout 22 kDa (little or no glycosylation present) to 38 kDa. Moreparticularly, SEQ ID NO:1 encodes Lu-ECAM-1-associated proteins ofapparent molecular size of about 38 kDa and of about 32 kDa. Further,these two LU-ECAM-1-associated proteins bind with Lu-ECAM-1 (amino acid1 to amino acid 799 of SEQ ID NO:2)in forming Lu-ECAM-1 complex. ThemCLCA, human CLCA1, and human CLCA2 were then cloned and sequenced usingthe Lu-ECAM-1 open reading frame as a probe.

In accordance with another embodiment of this invention, usingrecombinant techniques a nucleic acid molecule containing the nucleotidesequence encoding calcium activated chloride channel-adhesion moleculeis incorporated into an expression vector. The recombinant vector isintroduced into an appropriate host cell thereby directing theexpression of the sequence in that particular host cell. The expressionsystem, comprising the recombinant vector introduced into the host cell,can be used to produce recombinant CACC-AM, or associated proteins.According to the present invention, recombinant CACC-AM, a recombinantpolypeptide having CACC-AM activity, and/or recombinant CACC-AMassociated protein, can be purified by methods known in the artincluding ion-exchange chromatography, affinity chromatography, or otherchromatographic separation techniques.

Another embodiment of the present invention is a method for providingcalcium-activated chloride conductance channels to mammalian cells. Inmammalian cells in which the membrane chloride ion channels aredeficient in number or function (e.g., in airway epithelial cells ofcystic fibrosis patients), a method of providing to mammalian cells acalcium-activated chloride conductance channel, comprising CACC-AM or apolypeptide having CACC-AM activity, comprises administering directly tothe cells an expression vector. The expression vector contains a nucleicacid molecule operably linked to expression control sequences, whereinthe nucleic acid molecule encodes a CACC-AM, with the resultantexpression vector being introduced into the mammalian cell, and thecalcium-dependent chloride conductance produced in the mammalian cellscontaining the expression vector.

The bovine Lu-ECAM-1 complex appears to be expressed in lung, spleen,and aortic epithelial cells. The murine Lu-ECAM-1 complex appears to beexpressed in lung, trachea, spleen, mammary gland, intestine, uterus,epididymis, testis, pancreas, kidney, liver and skin. A first humanCLCA1 (hCLCA1) molecule (SEQ ID NO:28) appears to be expressed in smallintestine, and colon mucosa. A second human CLCA2 (hCLCA2) molecule (SEQID NO:32) appears to be expressed in trachea and mammary gland. A thirdhuman CLCA3 (hCLCA3) molecule (SEQ ID NO:30) appears to be expressed insmall intestine, trachea, mammary gland, stomach, bone marrow, spleen,lymph node, and peripheral blood leukocytes. That these variousmammalian proteins appear to be expressed in tissues which are affectedin cystic fibrosis may allow them to be used as chloride channels inaccordance with Example 8 herein.

For purposes of the description, the following embodiments illustratethe manner and process of making and using the invention and set forththe best mode contemplated by the inventor for carrying out theinvention, but are not to be construed as limiting.

EXAMPLE 1

This embodiment illustrates the molecular cloning of calcium activatedchloride channel-adhesion molecules. Lu-ECAM-1. A nucleic acid moleculeencoding Lu-ECAM-1 and Lu-ECAM-1-associated proteins according to thepresent invention can be obtained by preparing cDNA from total RNAisolated from a host cell expressing Lu-ECAM-1. To illustrate thisexample, total RNA was isolated from bovine aortic endothelial cells bythe guanidinium chloride procedure, and a Lu-ECAM-1 CDNA clone wasconstructed using nucleic acid amplification as summarized in FIG. 1.First, the N-terminal and internal amino acid sequences of a 38 kDaLu-ECAM-1-associated protein (SEQ ID NO:3) were used to designdegenerate primers for primary and nested polymerase chain reactionsusing the reverse-transcribed total RNA as template. Upstream primerscorresponded to nucleotide sequences encoding amino acids 685 to 693,and amino acids 698 to 705, of SEQ ID NO:3. Downstream antisense primerscorresponded to nucleotide sequences encoding amino acids 839 to 832,and amino acids 852 to 846, of SEQ ID NO:3. A product of approximately450 bp was amplified (illustrated in FIG. 1 as “P1”). From thesesequences, nondegenerate primers (SEQ ID NOs: 4 and 5) were designed,and the resultant amplification for 3′ sequences resulted in a productof approximately 750 bp (FIG. 1, “P2”). Nondegenerate primers (SEQ IDNOs: 6 and 7) were designed, and the resultant amplification for 5′sequences resulted in a product of approximately 1000 bp (FIG. 1, “P3”).To obtain the remaining 5′ sequences (FIG. 1, “24”) including a signalsequence and the ATG initiation codon, used was an internal primer (SEQID NO:8). To reconstitute the CDNA sequence from the amplified products(Pi-P4), the overlapping products were assembled into one open readingframe by an over-lap extension strategy using a high fidelity polymerasecombination. The result was clone 1 (FIG. 1) comprising 3.3 kb andencoding the amino acid sequence of SEQ ID NO:2. Hydrophilicity analysisrevealed six significant generally nonpolar regions. In particular, ahydrophobic sequence from amino acid 595 to amino acid 618 appears to bea transmembrane domain. Nine potential sites exist for asparagine-linkedglycosylation.

Using the primers to probe a lambda cDNA library, three additionalclones (clones 2, 3, and 4; FIG. 1) were identified and sequenced.Additional primers (SEQ ID NOs: 9 and 10) were used to obtain the 5′ endsequences. Clone 2, a 3.3 kb variant of clone 1, was identical to clone1 from nucleotide 252 to nucleotide 2438 of SEQ ID NO:1, but then thesequence diverged. The amino acid sequence deduced from clone 2 (SEQ IDNO:11) was identical to that of clone 1 up to amino acid 772 (of SEQ IDNO:2) followed by a glutamate and serine. Clone 3 was 2.8 kb variant ofclone 1. The amino acid sequence deduced from clone 3 (SEQ ID NO:12) wasidentical to that of clone 1 up to amino acid 772 (of SEQ ID NO:2),followed by an additional 28 amino acids. Clone 4, of 1.3 kb, appears toencode a truncated 321 amino acid (SEQ ID NO:13) variant of Lu-ECAM-1that may be secreted, and is identical in sequence to amino acids 1 to303 of SEQ ID NO:2, followed by 18 divergent amino acids. Anoligonucleotide probe (SEQ ID NO:14) synthesized from the unique 3′region of clone 1 was used to hybridize MRNA isolated from bovine aorticendothelial cells. The probe detected high molecular weight bands (6-10kb) in Northern blot analysis as well as the 3.3 kb band. However, theprobe did not hybridize to the 2.8 and 1.3 kb bands. These resultsindicate that the 38 kDa and 32 kDa proteins appear to be encoded onlyby the messages that also encode the 90 kDa protein.

This embodiment also illustrates that CACC-AM is conserved in mammalianspecies, and thus may serve the same or similar functions in mammalianspecies other than the ones disclosed herein. Conservation of the geneencoding CACC-AM was determined by multispecies genomic DNA (from human,green monkey, rat, mouse, dog, bovine, rabbit, chicken, and buddingyeast) blot with probes derived from various regions of the bovine cDNAsequence for Lu-ECAM-1. These probes hybridized to all mammalian speciesgenomic DNA, although the hybridization to rat DNA was comparativelyweak. No hybridization signal was detected for chicken DNA or yeast DNA.These results indicate that the gene(or variant sequence thereof)encoding Lu-ECAM-1 is highly conserved in mammalian evolution.

Accordingly, using similar methods and primer sequences for isolatingand sequencing of a nucleotide sequence encoding a bovine Lu-ECAM-1,various nucleotide sequences encoding other CACC-AMs maybe identified.

Mouse Calcium Activated Chloride Channel

As an illustration, a murine CACC/AM has been identified. A mouse lungcDNA library in lambda-gtll was screened with the open reading frame ofLu-ECAM-1 cDNA (EcoRl-BglII 2.4 kb fragment of the Lu-ECAM-1 cDNA) usinglow stringency hybridization conditions (hybridization at 65 C. in5×SSC, 5×Denhardt's solution and 0.2% SDS solution overnight withagitation; washing with 2×SSC followed by several washes in 0.2×SSC,0.2% SDS at room temperature for a total of 30 minutes). Positive phageswere purified and analyzed by Southern blot hybridization techniques.Standard sequencing techniques (eg. automatic sequencing techniques)were used to determine the sequence of the clones. The largest of theisolated CDNA was 2.2 kb in length. It lacked the 5′ end as determinedby sequence comparison with the known bovine homolog. A full lengthmouse Lu-ECAM-1 was constructed by amplification of the 5′ cDNA endsfrom a pool of mouse lung poly(A)+RNA (CLONTECH). A gene-specific primer(SEQ ID NO:35) was used to reverse transcribe the cDNA from mouse lungmRNA. A nested primer (SEQ ID NO:36) and a primer recognizing the 5′terminal tag were used to amplify the 5′ end of the cDNA by polymerasechain reaction. PCR products were cloned into an expressionvector(pGEM-3; Promega). A full length mouse mCLCAl was assembled byfusing the rapid amplification product clone with the 2.2 cDNA insert inan expression vector (pmlI site of pBluescript, Stratagene). Thus a 3.02kb long sequence (SEQ ID NO:33) encoding a polypeptide of 902 aminoacids (SEQ ID NO:34) was obtained.

Human CLCA1

In another illustration, a nucleic acid molecule encoding human calciumsensitive chloride channels was obtained from either the genomic libraryor a cDNA library. A human genomic library was screened with the ORE ofbovine Lu-ECAM-1 as probe using standard plaque hybridizationtechniques. Three positive clones of 4, 6, and 7 kb were isolated andsequenced, spanning a contiguous genomic fragment of 14 kb withinterspersed segments of 58 to 65% nucleotide identity to parts of theLu-ECAM-1 ORE. Since the regions of homology did not encode a contiguousopen reading frame and did not cover the entire Lu-ECAM-1 ORF theremaining parts of the gene were obtained by genomic walking usingnested PCR primers from each 5′ and 3′ end of the clones obtained byplaque hybridizations. Nested PCR conditions were 20 cycles for thefirst amplification step and 30 cycles for the second amplification withannealing temperatures of approximately 2° below the calculated meltingpoint of the primers and extension times of 5 mm per cycle. PCR productswere cloned into a vector (pGem-T, Promega) and sequenced. The fulllength gene was isolated and sequenced spanning 31,902 bp. The readingframe of the genomic sequence was determined according to its sequencehomology with bCLCA1, Lu-ECAM-1 and mCLCAl.

Using an RT-PCR based strategy, the CLCA1 cDNA was cloned and sequencedfrom small intestinal mRiNA. PCR primers (downstream primer SEQ IDNO:37, and upstream primer SEQ ID NO:38) flanking the ORE and containinglinkers with NotI restriction sites were generated and used to amplifythe 2745 bp ORF. RT-PCR was performed with 500 ng of human smallintestinal poly(A+) (CLONTECH). Reverse transcription was carried out at48° C. with Superscript RNase H-reverse transcriptase and PCR wasperformed with Pwo DNA polymerase (Boehringer). PCR conditions were asfollows: initial denaturation at 94° C. for 3 min followed by additionof DNA polymerase; 35 cycles of 94° C. for 50 s, 58° C. for 30 s, and72° C. for 2 min with a time increment of 3 s per cycle for eachextension step, followed by a final extension step of 72° C. for 8 min.Foe obtaining the untranslated region of CLCA1 mRNA, amplification ofthe 5′ and 3′ ends was carried out using primers SEQ ID NO:39 and SEQ IDNO:40 respectively. The resulting cDNA sequence (SEQ ID NO:27) comprises3007 bp and is identical to the genomic fragments with high sequencesimilarity to the previously cloned homolog. It contains a single ORF of2745 bp encoding a polypeptide of 914 amino acids (SEQ ID NO:28).

hCLCA2 cDNA

A human lung cDNA library (Clontech) was screened using Lu-ECAM-1 cDNAas probe as described above. Missing 5′ and 3′ ends of the isolated cDNAspecies were completed using RACE (Life Technologies). A single 3.6 kbcDNA species was identified and termed CLCA2. A sequence of 2970 bp isshown in SEQ ID NO: 31. The open reading frame of The nucleotidesequence encoding a polypeptide of 943 amino acids (SEQ ID NO:32) sharedhigh degrees of identity with those of Lu-ECAM-1 (86%), bCLCA1 (85%),mCLCAl (76%), and hCLCAI (63%)—FIG. 14.

hCLCA3 cDNA

A human spleen cDNA library packed in phage λgtll (Clontech)was screenedusing standard plaque hybridization protocols. The open reading frame(ORF) of the Lu-ECAM-1 cDNA was used as probe as described above. Phagecolony blots were hybridized and washed at low stringency conditions(hybridization: 55° C. overnight in 4×SSC standard hybridization bufferwithout formamide; two stringency washes with 2×SSC, 0.1% SDS at roomtemperature, and two washes with 1×SSC, 0.1% SDS at 40° C.). Afterexhaustive screening of the library (>7×10⁶ plaques), a single positivephage clone was plaque-purified, amplified, and subjected to DNApurification (Wizard Lambda Preps, Promega). The insert was cut outusing the EcoRI sites and cloned into pBluescript II SK (Stratagene).Automated sequencing with initial plasmid-derived primers followed byinternal gene-specific primers was performed by the Cornell UniversityDNA Sequencing Facility using dRhodamine Terminator Cycle Sequencing onan ABI Prism 377 DNA Sequencer (PE Applied Biosystems). Missing 5′ and3′ ends of the CDNA were isolated using the rapid amplification of CDNAends (RACE) technique (Life Technologies) and human spleen poly-A+RNA(Clontech) as template. The primers for amplification of 5′ end were SEQID NO:43 and SEQ ID NO:44, and the primers for 3′ end was SEQ ID NO:45.The resulting CDNA sequence of 3599 base paris (deposited in GenBankunder accession no. AF043976) was obtained. A sequence of 3418 bp isshown in SEQ ID NO:29, which encodes for a polypeptide of 1000 aminoacids (SEQ ID NO:30).

EXAMPLE 2

This example illustrates the proteins encoded by the cDNAs isolated inExample 1 and the relationship between CACC-AM and associated proteins.As an illustration, the relationship is between Lu-ECAM-1 and Lu-ECAM-1associated protein is demonstrated. Antigenic characterization wasperformed by generating anti-Lu-ECAM-1 antibodies, and testing theantibodies in Western blot analyses of bovine aortic endothelial cellextracts. Rats were immunized with either the 90 kDa band excised from apolyacrylamide gel and mixed with adjuvant, resulting in polyclonalantibody R4; or a 38 kDa band excised from a polyacrylamide gel andmixed with adjuvant, resulting in polyclonal antibody R41. Two peptides(SEQ ID NOs: 15 and 16) were synthesized, conjugated to KLH, and used toimmunize rabbits in forming polyclonal antibodies CU11 and CU8,respectively. Monoclonal antibody 6D3 has binding specificity toLu-ECAM-1 as described previously (Zhu et al., 1992, supra).

As shown in FIG. 2A, mAb 6D3 detected a 90 kDa component (Lu-ECAM-1) andtwo larger bands of approximately 120 kDa and 130 kDa (Lu-ECAM-1precursors); but not the 38 kDa or the 32 kDa components(Lu-ECAM-1-associated proteins). Likewise, polyclonal antibody (againstamino acid residues of SEQ ID NO:15) recognized only the 90 kDa, 120kDa, and 130 kDa components (FIG. 2A). In contrast, polyclonal antibodyCU19 (against amino acid residues 618 to 767 of SEQ ID NO:2) stronglydetected the 38 kDa and 32 kDa components, and the 120 kDa and 130 kDacomponents, but only weakly detected the 90 kDa component. These resultsare evidence that the initial translation products of the open readingframe in SEQ ID NO:1 are the 120 kDa and 130 kDa components, which arethen processed to yield the 90 kDa, 38 kDa, and 32 kDa components.

These results were confirmed with polyclonal antibodies R4 and R41. R4,a polyclonal anti-90 kDa protein antibody, detected the 90 kDa band, aswell as the 120 kDa and 130 kDa components; but not the 38 kDa, and 32kDa components (FIG. 2A). R41, a polyclonal anti-38 kDa proteinantibody, detected the 38 kDa and 32 kDa bands, as well as the 120 kDaand 130 kDa components; but not the 90 kDa component (FIG. 2A). Theseresults indicate that (a) the 38 kDa and 32 kDa bands are antigenicallyrelated; (b) the 120 kDa and 130 kDa bands are antigenically related;and (c) the 120 kDa and 130 kDa bands have sequence in common with boththe 90 kDa protein, and the 38 kDa and 32 kDa proteins. Treatment ofLu-ECAM-1 complex with Nglycosidase F reduced the 38 kDa and 32 kDacomponents to a common band of about 22 kDa, indicating the these twoproteins represent alternate glycoforms. N-glycosidase F treatmentreduced the 90 kDa protein to 77 kDa. The 77 kDa and 22 kDa productswould add up to the exact size of the initial translation product ofclone 1 before processing.

As shown in FIG. 2A, the 38 kpa and the 32 kDa components of theLu-ECAM-1 complex are not recognized by mAb 6D3 in SDS-PAGE and Westernblot analysis, suggesting that these components are likely noncovalentlycomplexed with the 90 kDa protein. The Lu-ECAM-1 complex is resistant todissociation by high salt, detergent, and EDTA, but readily dissociateswhen boiled in SDS in the presence or absence of reducing agents (e.g.,dithiothrietol). To visualize the Lu-ECAM-1 complex, and to determinewhether the proteins of the complex are associated intracellulary orextracellularly, the surface of bovine aortic endothelial cells wascross-linked. Confluent bovine aortic endothelial cells were surfacebiotinylated in the presence or absence of disuccinimidyl tartarate(DST), a reagent that restricts cross-linking to extracellular moietiesof proteins in close contact. DST dissolved in dimethyl sulf oxide wasadded to the cells in a final concentration of 1 mM. Cross-linking wascarried out at 4° C. with gentle shaking. The reactions were stopped byadding glycine to a final concentration of 50 mM. After quenching for 5minutes, the cells were lysed for 1 hour in lysis buffer. Lysates wereclarified by centrifugation, precipitated with mouse-IgG agarose beads,then immunoprecipitated with mAb 6D3. Immunoprecipitated proteins wereanalyzed by SDS-PAGE, transferred to nitrocellulose, and detected usingavidin-horseradish peroxidase and chemiluminescence. As shown in FIG.3A, immunoblots using either R4 (polyclonal anti-90 kDa proteinantibody) or R41 (polyclonal anti-38 kDa protein antibody) detected anovel band migrating at approximately 140 kDa (arrow, FIG. 3A), with aconcomitant reduction in intensities of the 90 kDa, 38 kDa, and 32 kDacomponents. All Lu-ECAM-1 complex components were biotinylated on bovineaortic endothelial cell surface. These results suggest that the Lu-ECAMcomplex is made up of either the 90 kDa and 38 kDa proteins complexed inan extracellular association, and/or the 90 kDa and 32 kDa proteinscomplexed in an extracellular association.

In another illustration of this embodiment, the mCLCAl protein wascharacterized. An in vitro transcription and translation system (TNT™,Promega) was used for the in vitro expression of the full length cDNA(SEQ ID NO:33). Canine microsomes were used to glycosylate the productof in vitro translation. In addition, HEK293 cells were transfected withthe cDNA of mCLCA1 using standard methods known to those skilled in theart (CaPO₄ or Lipfectamine, Life Technologies). Products were analyzedon SDS-PAGE gels. In addition, mCLCA1 cDNA was also used fortransfection of cells. Proteins prepared by standard in vitrotranslation techniques or from lysates of transfected HEK293 cells wereanalyzed on Western blotting by using rabbit polyclonal antibodiesagainst N-terminal (CU8) and the C-terminal region (CU2l) of Lu-ECAMpeptide. As shown in FIG. 6, protein bands of 130, 125, 90 kDa andtriplet bands of 32-38 kDa were detected in transfected cells. CU8reacted exclusively with the large sized bands of 90, 125 and 130 kDawhereas CU21 reacted with only the triplet of the smaller bands. Thisrecognition pattern is similar to that observed for Lu-ECAM-1 andsuggests that the ORF of mCLCA1 cDNA encodes a precursor protein,represented by alternate glycoforms of 125 and 130 kDa, that isposttranslationally processed into 90 kDa and 38/32 kDa components.

In another illustration of this embodiment, the hCLCA1 protein wascharacterized. The ORF of the hCLCA1 cDNA encodes a 914 amino acidprotein with a calculated molecular weight of 100.9 kDA. In vitrotranslation of human CLCA1 cDNA yielded a single protein ofapproximately 100 kDa, consistent with its calculated size (FIG. 7). Inthe presence of canine microsomes the Mr of the polypeptide shifted to125,000 indicating multiple glycosylations. Similar to Lu-ECAM-1 andmCLCA1, 37-40 kDa proteins were not detected in immunoblots of wholecell lysates but were coimmunoprecipitated with the 90 and 125 kDaprotein. To ascertain whether the 125 kDa hCLCA1 protein is processedinto 90 kDa and 30-40 kDa cleavage products in a manner similar toLu-ECAM-1, c-myc tags were inserted in five different hydrophilic siteswith high surface probability (m1-m5) and were overexpressed in HEK293cells (Cravchik et al., 1993, Gene 137:139-143). Immonoblots of wholecell lysates probed with anti-myc antibodies revealed proteins of 125and 90 kDa (FIG. 7b). However, immunoprecipitation of cell lysatesfollowing surface biotinylation indicated the presence of 37-41 kDaproteins similar to Lu-ECAM-1 and mCLCAl (FIG. 7c).

In another illustration of this embodiment, the human CLCA2protein wasanalyzed. The predicted size of the full length protein (104 kDa) isconsistent with the result of an in vitro translation assay yieldingprimary translation product of approximately 105 kDa (FIG. 8a) Toascertain whether the CLCA2 protein is cleaved into two subunits inmammalian cells as reported for other CLCAs, two constructs weregenerated with a c-myc tag within the amino or carboxy terminusrespectively as described by Cravchik et al., 1993, Gene 137:139-143)and transfected into HEK293 cells. Immunoblots of cell lysates probedwith anti-myc antibody identified an 86 kDa protein when the tag wasinserted near the amino terminus (m1) and a 34 kDa protein when the tagwas inserted near the amino terminus (m2)-FIG. 8b.

EXAMPLE 3

Tissue Distribution

This example illustrates the tissue distribution of CACC-AM. As anillustration, the distribution of Lu-ECAM-1/Lu-ECAM-1 complex in therespiratory tree, as demonstrated by immunohistochemistry. Tissuesections were probed with anti-Lu-ECAM-1 antibodies. Formalin-fixedsections of bovine trachea were first denatured by boiling for tenminutes in 4M urea in a microwave oven, then probed with polyclonalantibody R4 (raised against denatured Lu-ECAM-1). The sections were thenincubated with donkey anti-rat IgG and avidin-peroxidase conjugate. Theperoxidase conjugate was detected using diamino-benzidine as substrate,and then the slides were counterstained with hematoxylin. Lung sectionswere prepared and probed with mAb 6D3 as previously described (Zhu etal., 1993, mt. J. Cancer 53:68-633) except that a biotinylated secondaryantibody was used, followed by the avidin-peroxidase conjugate,diamino-benzidine as substrate, and counterstaining with hematoxylin.The immunohistochemical analyses revealed that Lu-ECAM-1/Lu-ECAM-1complex was expressed predominantly in endothelia of small tomedium-size venules of the lung, and in the respiratory epithelia ofbronchi and trachea. To confirm the distribution of expression ofLu-ECAM-1/Lu-ECAM-1 complex, and to distinguish it from that of thebovine epithelial chloride channel (“Ca-CC”) described recently(Cunningham et al., 1995, supra), nucleic acid amplification wasperformed using specific primers as described herein in Example 4.

Tissue distribution for other CACC-AMs of the present invention weredetermined by Northern blot analysis and RT-PCR. Human multiple tissueNorthern blots (Clontech) contained 2 μg poly-A+RNA per lane of thefollowing tissues: heart, brain, placenta, lung, liver, skeletal muscle,kidney, pancreas, spleen, thymus, prostate, testis, ovary, smallintestine, colon mucosa, peripheral blood leukocytes, stomach, thyroid,spinal cord, lymph node, trachea, adrenal gland, and bone marrow. Blotswere hybridized labeled fragments for respective cDNAs. To exclude crosshybridization of related family members, highly stringent washingconditions were employed following the hybridization (two washes with2×SSC, 0.1% SDS at 65° C. for 30 min, followed by two washes with0.2×SSC, 0.1% SDS at 65° C. for 30 min). RT-PCR was performed using theabove-mentioned conditions and primers to detect the cDNA fragments inpoly-A+RNA samples from human tissues. PCR products were analyzed on anethidium bromide stained agarose gel. To exclude amplification of aclosely related family member, the PCR products were cut out of the gel,cloned into the pGem-T vector, and partially sequenced. In all RT-PCRassays, negative controls were included with water instead of RNA astemplate in the reverse transcription. To control for RNA quality aswell as reverse transcription and PCR conditions, a fragment of EF-lamRNA was amplified as described.

A mouse multiple tissue Northern blot when probed with HindIII fragmentof mCLCAl ORF revealed the presence of a 3.1 kb transcript in brain andspleen and transcripts of 5 kb and 3.1 kb in heart, lung, liver, andkidney.

For human CLCA1, a single mRNA species of 3.3 kb was detected inNorthern blot hybridizations in small intestine and colon mucosa.Similar results were obtained with RT-PCR.

hCLCA2 mRNA was detected in trachea and mammary gland using the 2832 ORFof hCLCA1. While CLCA2 was not detected in the lung by Northern blothybridization, the more sensitive RT-PCR revealed its expression in lungin addition to trachea and mammary gland suggesting a significantlylower expression level in the lung.

No signals were detected in any of the tissues tested on Northern blotsusing the 2817 cDNA of hCLCA3. However, by RT-PCR a fragment of thehCLCA3 cDNA could be amplified form all tissues tested, i.e. spleen,lung, trachea, thymus and mammary gland.

EXAMPLE 4

This example demonstrates that Lu-ECAM-1 and the bovine epithelialchloride channel (“Ca-CC”) described recently by (Cunningham et al.,1995, J. Biol. Chem. 270:31016-31026) aredistinct molecules.

1. Genetic Similarity

Sequence alignment of the open reading frame of SEQ ID NO:1 with theCA-CC cDNA shows that the nucleotide sequences share 92% identity at theDNA level. Comparing the deduced amino acid sequence of Lu-ECAM-1 (SEQID NO:2) with that of CA-CC shows 88% identity at the amino acid level.However, the differences appear randomly distributed, and thus,Lu-ECAM-1 and CA-CC appear to represent products of different genes.

2. Subunit Differences

As shown in FIGS. 2A, 2B, 3A, and 3B, it is clear that the precursorLu-ECAM-1 is a protein with an apparent molecular size of either 120 kDaor 130 kDa. The precursor Lu-ECAM-1 gets processed to a 90 kDa Lu-ECAM-1protein, and to either a 38 kDa or 32 kDa Lu-ECAM-1-associated protein.In contrast, CA-CC is a 140 kDa multimeric complex that can be reducedto a band comprised of 38 kDa subunits in the presence of a reducingagent (Cunningham et al., 1995, supra). This difference in subunitstructure is further evidence that Lu-ECAM-1/Lu-ECAM-1 complex is aglycoprotein distinct from CA-CC.

3. Molecular Expression Differences

It is possible that immunohistochemical staining with polyclonalantibody to Lu-ECAM-1 could detect CA-CC if CA-CC shared across-reactive epitope with Lu-ECAM-1. To distinguish Lu-ECAM-1expression from CA-CC expression in tissues, reverse transcriptasepolymerase chain reaction was performed. Messenger RNA (500 ng) frombovine lung tissue, from bovine spleen tissue, from bovine trachealepithelium, and from cultured bovine aortic endothelial cells wasreverse-transcribed with random oligonucleotide primers and reversetranscriptase in a 20 μil reaction volume. Primers specific forLu-ECAM-1 sequences (primer pairs “L1”:SEQ ID NOS: 17 and 18, “L2”: SEQID NOs: 19 and 20), and primers specific for CA-CC sequences (primerpairs “T1”: SEQ ID NOs: 21 and 22, and “T2” SEQ ID NOs: 23 and 24) wereconfirmed for selectivity by control experiments with a Lu-ECAM-1 CDNAclone. Amplification was performed using 1 μl of the respective cDNAsubstrate for 35 cycles of amplification in a reaction volume of 50 [Liusing 0.5 units of thermostable DNA polymerase, 200, uM of each dNTP,1,5 mM MgCl₂, and 1 μM of the respective primer pair. The cyclingprotocol was 94° C. for 20 seconds, 55° C. for 10 seconds, and 72° C.for 10 seconds, with a time increment of 2 seconds per cycle forannealing and ° for 10 minutes. Aliquots (5 μl) of each amplificationreaction was fractionated on a 1.5% agarose gel, and stained withethidium bromide.

The calculated size for product amplified using primer pair L1 is 232bp; the calculated size for product amplified using primer pair L2 is218 bp; the calculated size for product amplified using primer pair T1is 231 bp; and the calculated size for product amplified using primerpair T2 is 218 bp. As shown in FIG. 4A, Lu-ECAM-1 is expressed in bovineaortic endothelial cells, lung tissue, and spleen, tissue, but not intracheal epithelium. In contrast, as shown in FIG. 4B, CA-CC isexpressed in lung tissue and tracheal epithelium, but not in bovineaortic endothelial cells nor spleen tissue. These results furthersupport that Lu-ECAM-1 and CA-CC are different molecular entities, withLu-ECAM-1 being expressed in venular endothelial cells, and CA-CC beingexpressed in tracheal and bronchial epithelial cells.

EXAMPLE 5

This embodiment illustrates that a nucleic acid molecule comprising anucleotide sequence encoding CACC-AM, or a variant sequence thereof, orencoding one or more CACC-AM associated proteins, can be inserted intovarious vectors including phage vectors and plasmids. Successfulexpression of the protein(s) requires that either the insert comprisingthe nucleotide sequence, or the vector itself, contain the necessaryelements for transcription and translation (expression control elements)which is compatible with, and recognized by the particular host systemused for expression. A variety of host systems may be utilized toexpress the recombinant protein(s), which include, but are not limitedto bacteria transformed with a bacteriophage vector, plasmid vector, orcosmid DNA; yeast containing yeast vectors; fungi containing fungalvectors; insect cell lines infected with virus (e.g. baculovirus); andmammalian cell lines transfected with plasmid or viral expressionvectors, or infected with recombinant virus (e.g. vaccinia virus,adenovirus, adeno-associated virus, retrovirus, etc.).

Using methods known in the art of molecular biology, including methodsdescribed above, various promoters and enhancers can be incorporatedinto the vector or the nucleic acid molecule encoding the recombinantprotease, to increase the expression of the recombinant protein(s),provided that this increased expression is compatible with (for example,non-toxic to) the particular host cell system used. The selection of thepromoter will depend on the expression system used. Promoters vary instrength, i.e. ability to facilitate transcription. Generally, for thepurpose of expressing a cloned gene, it is desirable to use a strongpromoter in order to obtain a high level of transcription of the gene orthe variant sequence and expression into the recombinant protein. Forexample, bacterial, phage, or plasmid promoters known in the art fromwhich a high level of transcription has been observed in a host cellsystem comprising E. coli include the lac promoter, trp promoter, tacpromoter, reca promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters, lacUV5, ompf, bla, lpp, and the like, may be used to providetranscription of the inserted DNA sequence encoding the recombinantprotein.

As known to those skilled in the art, such vectors for expression inmammalian cells can be selected from plasmids, viruses, andretroviruses. For a recent review of vectors useful in gene therapy, seeWeichselbaum and Kufe (1997, Lancet, 349:S10-S12). The features of avector which make it useful in the methods of the present inventioninclude that it have a selection marker for identifying vector which hasinserted therein the nucleotide sequence to be expressed; restrictionsites to facilitate cloning; and the ability to enter and/or replicatein mammalian cells. Examples of a preferred vector for the in vivointroduction of a recombinant vector into mammalian cells include, butare not limited to viral vectors. Virus-based vectors are one preferredvehicle as they infect cells in vivo, wherein during the infectionprocess the viral genetic material is transferred into the cells. Aretroviral vector, such as a plasmid containing AAV (Adeno-associatedvirus) sequences, has been described previously (see for exampleChatterjee et al., 1992, Science, 258:1485-1488; U.S. Pat. No.5,252,479, herein incorporated by reference). Examples of other vectorsfor the in vitro or in vivo introduction into mammalian cells includeretroviral vectors (Miller et al., 1989, BioTechriiq-ues 7:980-990;Korman et al., 1987, Proc. Nati. Acad. Sci. USA 84:2150-54), papovavirusepisomes (U.S. Pat. No. 5,624,820, herein incorporated by reference),and adenovirus vectors (U.S. Pat. No. 5,585,362, herein incorporated byreference). Promoters are known to those skilled in the art, and mayinclude viral or viral-like basal promoters like the 5V40 late promoter,the RSV promoter, the CMV immediate early promoter, and a VL30 promoter;and cellular promoters (See, e.g., Larsen et al., 1995, Nucleic AcidsRes. 23:1223-1230; Donis et al., 1993, BioTechniques 15:786-787; Dondaet al., 1993, Mol. Cell. Endocrinol. 90:R23-26; and Huper et al., 1992,In Vitro Cell Dev. Biol. 28A:730-734).

In one illustration of this embodiment, a nucleotide sequence comprisingclone 1 (SEQ ID NO:1) was placed under the control of atetracycline-regulated promoter in a commercially available plasmid(pTet-Splice; GIBCO). The construction was accomplished in two steps. Anamplified product was generated that corresponded to the 3′ end of clone1 CDNA (nucleotide 2391 to nucleotide 2780 of SEQ ID NO:1) using a 5′primer containing an EcoRI restriction site (SEQ ID NO:25) and a 3′primer containing a SpeI restriction site (SEQ ID NO:26). The cyclingprotocol included 93° C. for 35 seconds, 55° C. for 60 seconds, 72° C.for 3 minutes for 40 cycles followed by a 10 minute incubation at 72° C.using a thermostable DNA polymerase. The product was cleaved with EcoRIand SpeI, then cloned into corresponding restriction sites in theplasmid. The resultant plasmid was selected and then sequenced toconfirm absence of mutations. This recombinant plasmid was then cleavedwith EcoRI and BglII. To reconstitute the open reading frame encodingLu-ECAM-1, the 2.3 kb EcoRI/Bg1II fragment was excised from clone 3 andinserted into the plasmid. The resulting plasmid, pTet-Splice-Lu-ECAM-1,was then co-transfected into HEK293 cells with another plasmid(pTet-tTAK) that encodes a transcriptional activator specific for thepTet-Splice vector. Transfection was done using a transfection reagent(lipofectamine) according to the manufacturers instructions. Cells wereharvested 24 hours after the start of transfection. Immunoblot analysisof the cells using polyclonal R41 resulted in the detection ofrecombinant Lu-ECAM-1 precursor of 120 kDa, and recombinantLu-ECAM-1-associated protein of 38 kDa. When the cells were probed inimmunoblot with anti-peptide antibody CU8, detected was recombinantLu-ECAM-1 precursor of 120 kDa, and recombinant Lu-ECAM-1 of 90 kDa.

In another embodiment of the invention, mCLCA1 cDNA was cut from thepBluescript vector (Stratagene) with SacI and PvuI, blunt ended withKlenow Polymerase and inserted into the tetracycline sensitive mammalianexpression vector (pTet-splice, Life Technologies, Inc.) at the EcoRVsite. HEK293 cells were cotransfected with mCLCAl cDNA cloned into thepTet-splice along with a vector expressing a tetracycline activator(pTet-tTak) using standard transfection techniques well known to thoseskilled in the art and as described above (Lipofectamine, LifeTechnologies, Inc.). Cells were cotransfected with a reporter vector asdescribed above. In another illustration of this embodiment, humanCLCA1, HEK293 cells were transfected with either pcDNA 3.1 containingthe CLCA1 insert and a reporter vector (enhanced green fluorescentprotein, EGFP, CLONTECH) or the reporter vector alone. Transfection canbe carried out by standard techniques known to those skilled in the artincluding CaPO4 precipitation or Lipofectamine (Life Technologies).

For human CLCA2, HEK293 cells were transfected using Lipofectamine usingmanufacturer's instructions. For example, 5 ul lipid and 0.5 ul of CLCA2were cloned into pcDNA 3.1 per 35 mm well in a 2-3 hour incubation. Forexpression studies, the 2,832 bp CLCA2 ORF was PCR amplified from humantrachea poly-A⁺RNA (Clontech) following reverse transcription withSuperscript RNase H reverse transcriptase (Life Technologies) and randomhexamer priming. PCR was performed with Pwo DNA Polymerase (Boehringer;initial denaturation at 94° C. for 3 min, 35 cycles of 94° for 50 s, 58°C. for 30s, and 72° C. for 2 min with a time increment of 3 s per cyclefor each extension step (72° C.), followed by a final extension step of72° C. for 8 min). Primer sequences were (upstream primer: SEQ ID NO:41,downstream primer: SEQ ID NO:42 with NotI-linkers underlined). PCRproducts were gel purified, incubated with NotI, and cloned into theexpression vector pcDNA3.1 (Invitrogen). Four different PCR productswere sequenced to control for potential PCR-induced sequence errors.Cells were simultaneously cotransfected with a reporter vector asdescribed above. Chloride channel conductance activity was recordedafter allowing the cells to recover for 24 hours.

The 2817 bp fragment of the hCLCA3 cDNA cloned into pcDNA3.1 wassimultaneously transcribed and translated as described for the otherCACC-AMs. Samples were analyzed by 10% SDS-PAGE (5 μl of a 25 μlreaction), followed by drying of the gel and exposure to film for 8 h.Protease protection assays were performed as described to ascertainwhether hCLCA3 translation products were translocated into themicrosomes and thus entered the secretory pathway. In the presence ofmicrosomal membranes in vitro translated and ^(35S)-labeled wild typehCLCA3 was digested with Proteinase K (Sigma; 100 μg/ml) for 60 min onice with or without detergent present (0.5% Nonidet-P 40). The reactionwas stopped by adding phenylmethylsulfonyl fluoride and the productswere analyzed by 10% SDS-PAGE and exposure to film. To allow forimmunological detection of the translation products, three immunotaggedcDNA clones were constructed (m1 to m3) by inserting a partial sequenceof the human c-myc protein (EQKLISEEDL; SEQ ID NO: 47) into the aminotermini of the first (m1), the second (m2), or both (m3) ORFS.Generation of these constructs using overlap extension PCR and Pwo DNApolymerase (Boehringer) was as described. Correct sequences of theconstructs were verified by sequencing. Immunotagged DNA constructs wereeither in vitro translated as described above or transfected into 70%confluent human embryonic kidney (HEK) 293 or chinese hamster ovary(CHO) cells via the Lipofectamine Plus method (Life Technologies) Celllysates were harvested after 48 h, resolved via 10% SDS-PAGE, andelectroblotted onto nitrocellulose. Blots were probed withmouse-anti-human c-myc antibody 9E10 (1 μg/ml; Calbiochem) as primaryantibody, horseradish peroxidase-conjugated goat anti-mouse antibody(0.2 μg/ml) as secondary antibody, and developed using enhancedchemiluminescence (Amersham). Secretion of the recombinant hCLCA3protein into the culture supernatant was assayed by concentrating theconditioned medium (24 to 48 h after transfection) of HEK 293 or CHOcells transfected with construct m3 using ultrafiltration devices with amolecular cutoff at 10 kDa (Ultrafree-15, Biomax-l0 filter; Millipore;centrifugation at 2,000 g for 30 min at 4° C.).

EXAMPLE 6

This embodiment demonstrates that the CACC-AMs of the present inventioncan function as adhesion molecules. As an illustration, a recombinantLU-ECAM-1, encoded by a nucleic acid molecule according to the presentinvention, has unexpectedly improved biological activity. Recombinant(r) Lu-ECAM-1 and wild type (wt) Lu-ECAM-1 were compared in theiradhesion ability to lung-metastatic B16-Fl0 melanoma cells. Usinganti-Lu-ECAM-1 mAb 6D3, wtLu-ECAM-1 was purified from extracts of bovineaortic endothelial cells, and rLu-ECAM-1 was purified from extracts oftransfected HEK293 cells. The tumor cell adhesion assay was performed asdescribed previously (Zhu et al., 1992, supra). Briefly, 100 μg/ml inphosphate buffered saline of either wtLu-ECAM-1 or rLu-ECAM-1 was usedto coat wells of 96 plates overnight at 4° C. Wells were then washedwith tissue culture medium, and each well is seeded with a suspension oftissue culture medium and 2×104 tumor cells which had beenradio-labelled. After being spun onto the coated wells at 15 g for 1minute, and incubated for 10 minutes at 37° C., nonadherent tumor cellswere spun off at 150 g for 5 minutes. Adherent tumor cells were thendissolved in 1% SDS and counted in a liquid scintillation counter. Tumorcell attachment is recorded as the percent cells bound of the totalcells seeded. Inhibition of tumor cell adhesion is determined by firstincubating the Lu-ECAM-1 coated wells with mAb 6D3 (10 μg/ml) for 1 hourat room temperature before the tumor cells are added.

As shown in FIG. 5, recombinant Lu-ECAM-1 has unexpectedly improvedbiological activity (e.g., adhesive function to lung-metastatic tumorcells) as compared to wild type Lu-ECAM-1. More particularly, rLu-ECAM-1supported adhesion of 87of lung-metastatic tumor cells, whereaswtLu-ECAM-1 supported adhesion of only 43% of lung-metastatic tumorcells. Lung-metastatic tumor cell adhesion to wtLu-ECAM-1 was almostcompletely blocked by anti-Lu-ECAM-1 mAb 6D3, whereas lung-metastatictumor cell adhesion to rLu-ECAM-1 was only partially inhibited (66%) bythe concentration of anti-Lu-ECAM-1 mAb 6D3 used.

EXAMPLE 7

A comparison of the amino acid sequence of the CACC-AMs of the presentinvention is shown in FIG. 9. Sequence alignment and homology searcheswere carried out by using standard commercial software. For example,BLAST program was used for homology searches in existing data bases, andMegalign of the DNAStar package (Lasergene) was used for multiplesequence alignment. The sequence alignment of the four CACC-AMs of thepresent invention and the bovine CLCA (Cunningham et al. supra)indicates conservation throughout the entire length of the sequence,without the compartmentalization of more conserved domains. Nosignificant homologies to any other chloride channel proteins weredetected.

Table 1 illustrates a comparison of the size of the various mammalianLu-ECAM-1 proteins and Lu-ECAM-1 associated proteins as encoded-by therespective open reading frames.

TABLE 1 Total # of Species SEQ ID NO: Amino Acids Predicted Size bovine2 and 3 905 a.a. 90 kD, 32-28 kD human 28 914 a.a. 90 kD, 40 kD hCLCA1human 30 1000 a.a.  130 kD hCLCA3 (processing not known) human 32 943a.a. 130 kD hCLCA2 (processing not known) murine 34 902 a.a. 130 kD, 125kD mCLCA 90 kD, 32-38 kD

Table 2 is a comparison among the mammalian Lu-ECAM-1 family showingboth an approximated amino acid similarity and an approximated aminoacid identity (expressed as “similarity/identity”).

TABLE 2 bovine murine human human human (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NOs:2&3) NO:34) NO:28) NO:30) NO:32) bovine 100/100 81.3/70.867.4/52.4 85.7/77.4 63.7/49.8 (SEQ ID NOs:2&3) murine — 100/10067.5/52.7 80.9/69.5 62.8/48.4 mCLCA (SEQ ID NO:34) human — — 100/10065.3/51.4 62.3/44.7 hCLCA1 (SEQ ID NO:28) human — — — 100/100 62.1/48.2hCLCA3 (SEQ ID NO:30) human — — — — 100/100 hCLCA2 (SEQ ID NO:32)

Table 3 is a comparison among the mammalian Lu--ECAM-1 gene familyshowing approximated nucleic acid similarities (expressed in %).

TABLE 3 bovine murine human human human (SEQ ID (SEQ ID (SEQ ID (SED ID(SEQ ID NO:1) NO:33) NO:27) NO:29) NO:31) bovine 100 76.7 63.1 85.9 64.4(SEQ ID NO:1) murine — 100 62.6 76.1 61.2 (SEQ ID NO:33) human — — 10063.3 58.9 (SEQ ID NO:27) human — — — 100 62.6 (SEQ ID NO:29) human — — —— 100 (SEQ ID NO:31)

EXAMPLE 8

This embodiment illustrates that the full length cDNAs of the presentinvention encode calcium sensitive chloride channels. The various cDNAswere used for transfection of a cell line. For electrophysiologicalstudies, cells were also cotransfected with a reporter vector (pEGFP,CLONTECH). Cotransfection with a reporter vector allows for easyidentification of transfected cells by visualization under a fluorescentmicroscope. Whole cell recording was then carried out in the transfectedcells to determine the presence of calcium sensitive chloride channels.

Transfected cells were used for electrophysiological recording. Cellswere superfused with a bath solution containing 112 mM NMDG-Cl, 30 mMsucrose, 1 mM EGTA, 0.366 mM CaCl₂, 2 mM MgCl₂, 5 mMN-2-hydroxy-xyethanylpiperazine-N-2-ethanesulfonic acid. Whole cellchannel activity was recorded in transfected cells by using borosilicateglass electrodes (tip resistance 4-9 M ohms) filled with the bathsolution. Recordings were carried out in the presence or absence of acalcium channel inhibitors (DIDS, niflumic acid and DTT). To determinethe effect of ionomycin on channel activity, electrodes filled withstandard bath solution containing either 5 mM ATP and 1 mM EGTA in thepresence of low intracellular calcium. After gigaohm seal formation,cells were clamped at +20 mV. Whole cell currents were recorded at roomtemperature, sampled at 5-10 kHz and filtered at 1-2 kHz. The I-Vrelationship was determined using 300 ms voltage steps from a holdingpotential of +20 mV to potentials from −100 to +100 mV at 10 mVintervals. To normalize measured membrane currents to membrane currentsto membrane capacitance, the capacitive current transient recorded inresponse to a 10 mV hyperpolarizing pulse was integrated and divided bythe given voltage to give total membrane capacitance (C_(m)) for eachcell.

As shown in FIG. 9, expression of mCLCA₁ in HEK293 cells was associatedwith the appearance of a novel Ca2⁺sensitive Cl-channel as determined bywhole cell recordings in the presence and absence of the Ca2⁺ionophoreionomycin (2 uM). As shown in FIG. 9b, at low intracellular freeCa2+concentrations, the basal current at +100 mV in mCLCA1-transfectedcells was 2.05±1.09 pA/pF. With ionomycin the current increased to10.23±3.46 pA/pF. No significant effect of these manipulations was seenin non-transfected or control-transfected cells. Basal currents in thepresence of 2 mM Ca2+in transfected cells averaged 12.01±6.31 pA/pF.Perfusion of 300 uM DIDS reduced the current to 1.84 ±0.96. A similareffect was seen with NFA and DTT. These results indicate that theexpression of mCLCA1 in HEK293 cells is associated with the appearanceof a Ca2+sensitive chloride conductance. Under whole cell conditions,the current was outwardly rectified and inhibited by the anion channelblockers DIDS and NFA as well as the reducing agent DTT. This data issummarized in FIG. 10.

Whole cell recording of cells transfected with hCLCA1 cDNA demonstratedthe induction of calcium sensitive chloride channels (FIG. 11). Externalperfusion of ionomycin (2 uM) was associated with an increase in themaximally activated current at +100 mV from 0.65 to 11.06 pA/pF. Thecurrent voltage relationship was outwardly rectified and reversed at 0mV under symmetrical recording conditions. No effect of ionomycin wasobserved on non-transfected cells or control transfected cells. Additionof DIDS, DTT or niflumic acid reduced the currents to 1.63, 1.67 and2.07 pA/pF respectively Cell attached patch recordings of singlechannels confirmed the presence of calcium sensitive anion channel (datanot shown). This data is summarized in FIG. 12.

Whole cell recordings of hCLCA2 transfected HEK293 cells exhibited sslightly outwardly rectifying current/voltage relationship that wasabsent from control cells (transfected with vector alone; FIG. 13). Thiscurrent was sensitive to DIDS (300 uM), DTT (2 mM), niflumic acid (100uM), and tamoxifen (10 uM). When the pipet solution contained lowCa2=(about 25 nM) with 2 mM Ca2+in the bath, perfusion of theCa2+ionophore ionomycin (4 uM) through the bath also activated thecurrent (FIG. 13e).

These results indicate that the expression of CACC/AM moleculesdisclosed herein and their variants is associated with the appearance ofcalcium sensitive chloride channels.

EXAMPLE 9

This embodiment illustrates uses of the sequences according to thepresent invention. In one embodiment of the present invention, anindividual having a primary tumor having lung-metastatic capabilities istreated with an anti-adhesion therapy comprising administering to theindividual a therapeutically effective amount of a compositioncomprising either antibody raised to rLu-ECAM-1 or recombinant Lu-ECAM-1complex, or a vector for expressing a soluble form of rLu-ECAM-1 orrLu-ECAM-1 complex which can then bind to the lung-metastatic tumorcells. Either composition may function to prevent lung-metastatic tumorcell adhesion to the lung venule endothelial cells, thereby preventingcolonization by the metastatic tumor cells. As known to those skilled inthe art, an effective amount of a therapeutic composition may depend onthe route of administration (e.g., intravenous or other route known inthe art), and physiological factors including the age, size, and rate ofmetabolism of the individual to be treated.

Another embodiment of the present invention is a method for providingcalcium-dependent chloride conductance channels to mammalian cells.Recombinant Lu-ECAM-1 or rLu-ECAM-1 complex may form a chloride channelwhich may affect chloride secretion, and hence fluid secretion, from thecell. It may be that the chloride ion channel is coupled to the adhesionprocess involving the binding of Lu-ECAM-1 to a ligand, as similarlyobserved for the adherence and growth of lymphatic endothelial cells(Martin et al., 1996, supra). Thus, in mammalian cells in which themembrane chloride ion channels are deficient in number or function(e.g., in airway epithelial cells of cystic fibrosis patients), a methodof providing to mammalian cells a calcium-dependent chloride conductancechannel, rLu-ECAM-1 or rLu-ECAM-1 complex, comprises administeringdirectly to the lung endothelial and/or epithelial cells (in vitro or invivo) an expression vector. The expression vector contains a nucleicacid molecule(or a variant thereof) operably linked to expressioncontrol sequences, wherein the nucleic acid molecule encodes eitherrLu-ECAM-1 or rLu-ECAM-1 complex, with the resultant expression vectorbeing introduced into the mammalian cell, and a functionalcalcium-dependent chloride conductance channel produced in the mammaliancells which contain the expression vector. The cells targeted forchloride conductance channel production may include airway cellsselected from the group consisting of tracheal, bronchial or lung cells.If the cells are transfected in vitro, the transfected cells may then beintroduced in vivo into the area of the lungs of the individual which isdeficient in chloride channel function.

Having described the preferred embodiments of the present invention, itwill be apparent to one of ordinary skill in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 47 <210> SEQ ID NO 1 <211> LENGTH: 3317<212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: sequence encoding Lu-ECAM-1  #and Lu-ECAM-1      associated protein from bovine endot #helial cells<400> SEQUENCE: 1ggattccagg gtctccagca ttgcctgaat ctggatgtag gtttactgta  #              50 acatgtgcaa aa atg gtg ctc tgt ctg aat gtt att# ctg ttc cta act         98 ttg cat ctc ttg cct gga atg aaa agt tca at#g gta aat ttg att          14 #3aac aat ggg tat gat ggc att gtc att gca at#t aac ccc agt gtg          18 #8cca gaa gat gaa aaa ctc att gaa aac ata aa#g gaa atg gta act          23 #3gaa gct tct act tac ctg ttt cat gcc acc aa#a cga aga gtt tat          27 #8ttc agg aat gtg agc att tta att cca atg ac#c tgg aaa tca aaa          32 #3tct gag tac ttc ata cca aaa caa gaa tca ta#t gac cag gca gat          36 #8gtc ata gtt gct aat ccc tat cta aaa tat gg#a gat gat ccc tat          41 #3aca ctt caa tat gga agg tgt gga gaa aaa gg#a aaa tat ata cat          45 #8ttt act cca aac ttc ttg ttg act aat aat tt#c cac atc tat ggg          50 #3tcc cga ggc aga gta ttt gtc cat gag tgg gc#c cat ctc cgc tgg          54 #8gga ata ttt gat gag tat aat gtg gac cag cc#a ttc tat att tcc          59 #3aga aag aac act att gaa gca aca aga tgt tc#a act cat att act          63 #8ggt att aat gtg gtt ttc aag aaa tgc cct gg#a ggc agc tgt ata          68 #3aca agt cta tgc aga cgt gac tca cag aca gg#g ctg tat gaa gca          72 #8aaa tgt aca ttc ctt cca aaa aaa tcc cag ac#t gca aag gaa tcc          77 #3att atg ttt atg cca agt ctc cat tct gtg ac#t gaa ttt tgt aca          81 #8gaa aaa aca cac aat aca gaa gct cca aac ct#a caa aac aaa atg          86 #3tgc aat ggc aaa agc aca tgg gat gta atc at#g aac tct gtt gac          90 #8ttt cag aat aca tct ccc atg aca gaa atg aa#t cca ccg act cat          95 #3cct aca ttt tca ttg ctc aag tcc aaa cag cg#g gta gtc tgt ttg          99 #8gta ctt gat aaa tct gga agc atg tct gca ga#a gac cgt ctc ttt         1043caa atg aat caa gca gca gaa cta tac ttg at#t caa gtt att gaa         1088aag gga tct tta gtt ggg atg gtt aca ttt ga#c agt gtt gct gaa         1133atc caa aat cat cta aca aga ata act gat ga#t aat gtt tac caa         1178aag atc acc gca aaa ctg cct caa gta gct aa#t ggt gga act tca         1223att tgt aga ggg ctc aaa gca gga ttc cag gc#a att atc cac agt         1268gac cag agt act tct ggt tct gaa atc ata ct#a tta act gat ggg         1313gaa gat aat gaa ata aat tca tgc ttt gag ga#t gta aaa cga agt         1358ggt gca atc atc cac acc att gct ctg gga cc#c tct gct gcc aaa         1403gaa ctg gag aca ttg tca aat atg aca gga gg#a tat cgt ttt ttt         1448gcc aat aaa gac ata act ggc ctt act aat gc#t ttc agt aga att         1493tca tct aga agt gga agc atc act cag cag gc#t att cag ttg gaa         1538agc aaa gcc ttg aaa att aca gga agg aaa ag#a gta aac ggc aca         1583gtg cct gta gac agt aca gtt gga aat gac ac#t ttc ttt gtt gtc         1628aca tgg aca ata caa aaa cca gaa att gtt ct#c caa gat cca aaa         1673gga aag aaa tat aaa acc tcg gat ttc aaa ga#a gat aag tta aat         1718att cga tct gct cgt ctg caa ata cct ggt at#t gca gag aca ggt         1763act tgg act tac agc ctt cta aat aat cat gc#c agc tct caa atg         1808cta aca gtg aca gtg acc act cga gca aga ag#t cct act ata ccc         1853cca gta att gca aca gct cac atg agt caa ca#t aca gca cat tat         1898cct agc cca atg att gtt tat gca caa gtc ag#t caa ggg ttt ttg         1943cct gta ctg gga atc agt gta ata gcc att at#a gaa acc gaa gat         1988gga cat caa gta aca ttg gag ctc tgg gac aa#t ggt gca ggt cgt         2033gat act gtc aag aat gat ggc atc tac tca ag#a tac ttt aca gat         2078tac tat gga aat ggt aga tac agt tta aaa gt#a cat gca cag gca         2123aga aac aac acg gct agg cta aat tta aga ca#a cca cag aac aaa         2168gtt cta tat gtt cca ggc tac gtt gaa aac gg#t aaa att ata ctg         2213aac cca ccc aga cct gaa gtc aaa gat gac ct#g gca aaa gct aaa         2258ata gaa gac ttt agc aga cta acc tct gga gg#g tca ttt act gta         2303tca gga gct cct cct cct ggt aat cac cct tc#t gtg ttc cca ccc         2348agt aaa att aca gat ctt gag gct aag ttc aa#a gaa gat tat att         2393caa ctt tca tgg aca gcc cct ggc aat gtc ct#a gat aaa gga aaa         2438gcc aac agc tac att ata aga ata agt aag ag#t ttc atg gat cgt         2483caa gaa gat ttt gac aat gcg act tta gtg aa#t act tct aat cta         2528ata cct aag gag gcc gga tca aaa gaa aat tt#t gaa ttt aag cca         2573gaa cat ttt aga gta gaa aat ggc acc aaa tt#c tat att tca gtc         2618caa gcc atc aac gaa gcc aat ctc atc tca ga#g gtt tct cac att         2663gta caa gca atc aaa ttt att cct cta cca ga#a gac agt gtc cat         2708gat ctg ggt acc aag att tct gaa atc act ct#g gca att tta gga         2753tta cca atg att ttc tct gta ttt taaactagga at#tgtgtcag               279 #7cactgataac caatgttata catagttggt acacatttat ttaggattta  #            2847attcgctatt ttcttgttct tcagtagcta aattgtgtcc aaccttgcga  #            2897ctgcaggact gcagcatgcc aggtttccct gtccatcacc aactcccaga  #            2947gcttgctcaa atccatgttc atttgagtca gtaatgctaa ctatctcatc  #            2997ctctactgcc ctcttctctg tttaccttca atctttcccc agcattagga  #            3047tcttttccaa tgagtcagct cttagcatcg ggtggccaaa atattggcat  #            3097tttcagcaac agttcttcaa atgaaatatc cagggtgatt ttctttagga  #            3147tagactggtg actgacagtt caagggacac tctggagtct tctccagcac  #            3197cgcaccgcag tttgaaagaa ccagttcttt ggtactcagc cttctttata  #            3247gtccaatgct cacatctatc atgactcctg gaaaaaccat agctttgaga  #            3297 aatggatctt tgttgggaaa             #                  #                 331 #7 <210> SEQ ID NO 2 <211> LENGTH: 905<212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Lu-ECAM-1 precursor from  #bovine endothelial      cells <400> SEQUENCE: 2 Met Val Leu Cys Leu Asn Val Ile Leu Phe Le#u Thr Leu His Leu     -20               #   -15               #   -10Leu Pro Gly Met Lys Ser Ser Met Val Asn Le #u Ile Asn Asn Gly    -5               #     1              #  5Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp10                   # 15                  # 20Glu Lys Leu Ile Glu Asn Ile Lys Glu Met Va #l Thr Glu Ala Ser25                   # 30                  # 35Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Va #l Tyr Phe Arg Asn40                   # 45                  # 50Val Ser Ile Leu Ile Pro Met Thr Trp Lys Se #r Lys Ser Glu Tyr55                   # 60                  # 65Phe Ile Pro Lys Gln Glu Ser Tyr Asp Gln Al #a Asp Val Ile Val70                   # 75                  # 80Ala Asn Pro Tyr Leu Lys Tyr Gly Asp Asp Pr #o Tyr Thr Leu Gln85                   # 90                  # 95Tyr Gly Arg Cys Gly Glu Lys Gly Lys Tyr Il #e His Phe Thr Pro100                 1 #05                 1 #10Asn Phe Leu Leu Thr Asn Asn Phe His Ile Ty #r Gly Ser Arg Gly115                 1 #20                 1 #25Arg Val Phe Val His Glu Trp Ala His Leu Ar #g Trp Gly Ile Phe130                 1 #35                 1 #40Asp Glu Tyr Asn Val Asp Gln Pro Phe Tyr Il #e Ser Arg Lys Asn145                 1 #50                 1 #55Thr Ile Glu Ala Thr Arg Cys Ser Thr His Il #e Thr Gly Ile Asn160                 1 #65                 1 #70Val Val Phe Lys Lys Cys Pro Gly Gly Ser Cy #s Ile Thr Ser Leu175                 1 #80                 1 #85Cys Arg Arg Asp Ser Gln Thr Gly Leu Tyr Gl #u Ala Lys Cys Thr190                 1 #95                 2 #00Phe Leu Pro Lys Lys Ser Gln Thr Ala Lys Gl #u Ser Ile Met Phe205                 2 #10                 2 #15Met Pro Ser Leu His Ser Val Thr Glu Phe Cy #s Thr Glu Lys Thr220                 2 #25                 2 #30His Asn Thr Glu Ala Pro Asn Leu Gln Asn Ly #s Met Cys Asn Gly235                 2 #40                 2 #45Lys Ser Thr Trp Asp Val Ile Met Asn Ser Va #l Asp Phe Gln Asn250                 2 #55                 2 #60Thr Ser Pro Met Thr Glu Met Asn Pro Pro Th #r His Pro Thr Phe265                 2 #70                 2 #75Ser Leu Leu Lys Ser Lys Gln Arg Val Val Cy #s Leu Val Leu Asp280                 2 #85                 2 #90Lys Ser Gly Ser Met Ser Ala Glu Asp Arg Le #u Phe Gln Met Asn295                 3 #00                 3 #05Gln Ala Ala Glu Leu Tyr Leu Ile Gln Val Il #e Glu Lys Gly Ser310                 3 #15                 3 #20Leu Val Gly Met Val Thr Phe Asp Ser Val Al #a Glu Ile Gln Asn325                 3 #30                 3 #35His Leu Thr Arg Ile Thr Asp Asp Asn Val Ty #r Gln Lys Ile Thr340                 3 #45                 3 #50Ala Lys Leu Pro Gln Val Ala Asn Gly Gly Th #r Ser Ile Cys Arg355                 3 #60                 3 #65Gly Leu Lys Ala Gly Phe Gln Ala Ile Ile Hi #s Ser Asp Gln Ser370                 3 #75                 3 #80Thr Ser Gly Ser Glu Ile Ile Leu Leu Thr As #p Gly Glu Asp Asn385                 3 #90                 3 #95Glu Ile Asn Ser Cys Phe Glu Asp Val Lys Ar #g Ser Gly Ala Ile400                 4 #05                 4 #10Ile His Thr Ile Ala Leu Gly Pro Ser Ala Al #a Lys Glu Leu Glu415                 4 #20                 4 #25Thr Lys Ser Asn Met Thr Gly Gly Tyr Arg Ph #e Phe Ala Asn Lys430                 4 #35                 4 #40Asp Ile Thr Gly Leu Thr Asn Ala Phe Ser Ar #g Ile Ser Ser Arg445                 4 #50                 4 #55Ser Gly Ser Ile Thr Gln Gln Ala Ile Gln Le #u Glu Ser Lys Ala460                 4 #65                 4 #70Leu Lys Ile Thr Gly Arg Lys Arg Val Asn Gl #y Thr Val Pro Val475                 4 #80                 4 #85Asp Ser Thr Val Gly Asn Asp Thr Phe Phe Va #l Val Thr Trp Thr490                 4 #95                 5 #00Ile Gln Lys Pro Glu Ile Val Leu Gln Asp Pr #o Lys Gly Lys Lys505                 5 #10                 5 #15Tyr Lys Thr Ser Asp Phe Lys Glu Asp Lys Le #u Asn Ile Arg Ser520                 5 #25                 5 #30Ala Arg Leu Gln Ile Pro Gly Ile Ala Glu Th #r Gly Thr Trp Thr535                 5 #40                 5 #45Tyr Ser Leu Leu Asn Asn His Ala Ser Ser Gl #n Met Leu Thr Val550                 5 #55                 5 #60Thr Val Thr Thr Arg Ala Arg Ser Pro Thr Il #e Pro Pro Val Ile565                 5 #70                 5 #75Ala Thr Ala His Met Ser Gln His Thr Ala Hi #s Tyr Pro Ser Pro580                 5 #85                 5 #90Met Ile Val Tyr Ala Gln Val Ser Gln Gly Ph #e Leu Pro Val Leu595                 6 #00                 6 #05Gly Ile Ser Val Ile Ala Ile Ile Glu Thr Gl #u Asp Gly His Gln610                 6 #15                 6 #20Val Thr Leu Glu Leu Trp Asp Asn Gly Ala Gl #y Arg Asp Thr Val625                 6 #30                 6 #35Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe Th #r Asp Tyr Tyr Gly640                 6 #45                 6 #50Asn Gly Arg Tyr Ser Leu Lys Val His Ala Gl #n Ala Arg Asn Asn655                 6 #60                 6 #65Thr Ala Arg Leu Asn Leu Arg Gln Pro Gln As #n Lys Val Leu Tyr670                 6 #75                 6 #80Val Pro Gly Tyr Val Glu Asn Gly Lys Ile Il #e Leu Asn Pro Pro685                 6 #90                 6 #95Arg Pro Glu Val Lys Asp Asp Leu Ala Lys Al #a Lys Ile Glu Asp700                 7 #05                 7 #10Phe Ser Arg Leu Thr Ser Gly Gly Ser Phe Th #r Val Ser Gly Ala715                 7 #20                 7 #25Pro Pro Pro Gly Asn His Pro Ser Val Phe Pr #o Pro Ser Lys Ile730                 7 #35                 7 #40Thr Asp Leu Glu Ala Lys Phe Lys Glu Asp Ty #r Ile Gln Leu Ser745                 7 #50                 7 #55Trp Thr Ala Pro Gly Asn Val Leu Asp Lys Gl #y Lys Ala Asn Ser760                 7 #65                 7 #70Tyr Ile Ile Arg Ile Ser Lys Ser Phe Met As #p Arg Gln Glu Asp775                 7 #80                 7 #85Phe Asp Asn Ala Thr Leu Val Asn Thr Ser As #n Leu Ile Pro Lys790                 7 #95                 8 #00Glu Ala Gly Ser Lys Glu Asn Phe Glu Phe Ly #s Pro Glu His Phe805                 8 #10                 8 #15Arg Val Glu Asn Gly Thr Lys Phe Tyr Ile Se #r Val Gln Ala Ile820                 8 #25                 8 #30Asn Glu Ala Asn Leu Ile Ser Glu Val Ser Hi #s Ile Val Gln Ala835                 8 #40                 8 #45Ile Lys Phe Ile Pro Leu Pro Glu Asp Ser Va #l His Asp Leu Gly850                 8 #55                 8 #60Thr Lys Ile Ser Glu Ile Thr Leu Ala Ile Le #u Gly Leu Pro Met865                 8 #70                 8 #75 Ile Phe Ser Val Phe880             884 <210> SEQ ID NO 3 <211> LENGTH: 203 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Lu-ECAM-1 associated protein # from bovine      endothelial cells <400> SEQUENCE: 3Val Leu Tyr Val Pro Gly Tyr Val Glu Asn Gl #y Lys Ile Ile Leu  1              5   #                 10  #                15Asn Pro Pro Arg Pro Glu Val Lys Asp Asp Le #u Ala Lys Ala Lys                 20  #                 25  #                30Ile Glu Asp Phe Ser Arg Leu Thr Ser Gly Gl #y Ser Phe Thr Val                 35  #                 40  #                45Ser Gly Ala Pro Pro Pro Gly Asn His Pro Se #r Val Phe Pro Pro                 50  #                 55  #                60Ser Lys Ile Thr Asp Leu Glu Ala Lys Phe Ly #s Glu Asp Tyr Ile                 65  #                 70  #                75Gln Leu Ser Trp Thr Ala Pro Gly Asn Val Le #u Asp Lys Gly Lys                 80  #                 85  #                90Ala Asn Ser Tyr Ile Ile Arg Ile Ser Lys Se #r Phe Met Asp Arg                 95  #                100  #                105Gln Glu Asp Phe Asp Asn Ala Thr Leu Val As #n Thr Ser Asn Leu                110   #               115   #               120Ile Pro Lys Glu Ala Gly Ser Lys Glu Asn Ph #e Glu Phe Lys Pro                125   #               130   #               135Glu His Phe Arg Val Glu Asn Gly Thr Lys Ph #e Tyr Ile Ser Val                140   #               145   #               150Gln Ala Ile Asn Glu Ala Asn Leu Ile Ser Gl #u Val Ser His Ile                155   #               160   #               165Val Gln Ala Ile Lys Phe Ile Pro Leu Pro Gl #u Asp Ser Val His                170   #               175   #               180Asp Leu Gly Thr Lys Ile Ser Glu Ile Thr Le #u Ala Ile Leu Gly                185   #               190   #               195Leu Pro Met Ile Phe Ser Val Phe                 200   #       203<210> SEQ ID NO 4 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 4aatttaagcc agaacatttt agagta           #                  #              26 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 5gaaaatggca ccaaattcta tat            #                  #                23 <210> SEQ ID NO 6 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 6atatagaatt tggtgccatt ttc            #                  #                23 <210> SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 7tagaagtatt cactaaagt              #                  #                   # 19 <210> SEQ ID NO 8 <211> LENGTH: 28<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 8tactgtctac aggcactgtg ccgtttac          #                  #             28 <210> SEQ ID NO 9 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 9ggaatatttg atgagtat              #                   #                  #  18 <210> SEQ ID NO 10 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 10attcatttga aagagacg              #                   #                  #  18 <210> SEQ ID NO 11 <211> LENGTH: 795 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Variant of Lu-ECAM-1 from # bovine endothelial      cells <400> SEQUENCE: 11Met Val Leu Cys Leu Asn Val Ile Leu Phe Le #u Thr Leu His Leu    -20               #   -15               #   -10Leu Pro Gly Met Lys Ser Ser Met Val Asn Le #u Ile Asn Asn Gly    -5               #     1              #  5Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp10                   # 15                  # 20Glu Lys Leu Ile Glu Asn Ile Lys Glu Met Va #l Thr Glu Ala Ser25                   # 30                  # 35Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Va #l Tyr Phe Arg Asn40                   # 45                  # 50Val Ser Ile Leu Ile Pro Met Thr Trp Lys Se #r Lys Ser Glu Tyr55                   # 60                  # 65Phe Ile Pro Lys Gln Glu Ser Tyr Asp Gln Al #a Asp Val Ile Val70                   # 75                  # 80Ala Asn Pro Tyr Leu Lys Tyr Gly Asp Asp Pr #o Tyr Thr Leu Gln85                   # 90                  # 95Tyr Gly Arg Cys Gly Glu Lys Gly Lys Tyr Il #e His Phe Thr Pro100                 1 #05                 1 #10Asn Phe Leu Leu Thr Asn Asn Phe His Ile Ty #r Gly Ser Arg Gly115                 1 #20                 1 #25Arg Val Phe Val His Glu Trp Ala His Leu Ar #g Trp Gly Ile Phe130                 1 #35                 1 #40Asp Glu Tyr Asn Val Asp Gln Pro Phe Tyr Il #e Ser Arg Lys Asn145                 1 #50                 1 #55Thr Ile Glu Ala Thr Arg Cys Ser Thr His Il #e Thr Gly Ile Asn160                 1 #65                 1 #70Val Val Phe Lys Lys Cys Pro Gly Gly Ser Cy #s Ile Thr Ser Leu175                 1 #80                 1 #85Cys Arg Arg Asp Ser Gln Thr Gly Leu Tyr Gl #u Ala Lys Cys Thr190                 1 #95                 2 #00Phe Leu Pro Lys Lys Ser Gln Thr Ala Lys Gl #u Ser Ile Met Phe205                 2 #10                 2 #15Met Pro Ser Leu His Ser Val Thr Glu Phe Cy #s Thr Glu Lys Thr220                 2 #25                 2 #30His Asn Thr Glu Ala Pro Asn Leu Gln Asn Ly #s Met Cys Asn Gly235                 2 #40                 2 #45Lys Ser Thr Trp Asp Val Ile Met Asn Ser Va #l Asp Phe Gln Asn250                 2 #55                 2 #60Thr Ser Pro Met Thr Glu Met Asn Pro Pro Th #r His Pro Thr Phe265                 2 #70                 2 #75Ser Leu Leu Lys Ser Lys Gln Arg Val Val Cy #s Leu Val Leu Asp280                 2 #85                 2 #90Lys Ser Gly Ser Met Ser Ala Glu Asp Arg Le #u Phe Gln Met Asn295                 3 #00                 3 #05Gln Ala Ala Glu Leu Tyr Leu Ile Gln Val Il #e Glu Lys Gly Ser310                 3 #15                 3 #20Leu Val Gly Met Val Thr Phe Asp Ser Val Al #a Glu Ile Gln Asn325                 3 #30                 3 #35His Leu Thr Arg Ile Thr Asp Asp Asn Val Ty #r Gln Lys Ile Thr340                 3 #45                 3 #50Ala Lys Leu Pro Gln Val Ala Asn Gly Gly Th #r Ser Ile Cys Arg355                 3 #60                 3 #65Gly Leu Lys Ala Gly Phe Gln Ala Ile Ile Hi #s Ser Asp Gln Ser370                 3 #75                 3 #80Thr Ser Gly Ser Glu Ile Ile Leu Leu Thr As #p Gly Glu Asp Asn385                 3 #90                 3 #95Glu Ile Asn Ser Cys Phe Glu Asp Val Lys Ar #g Ser Gly Ala Ile400                 4 #05                 4 #10Ile His Thr Ile Ala Leu Gly Pro Ser Ala Al #a Lys Glu Leu Glu415                 4 #20                 4 #25Thr Lys Ser Asn Met Thr Gly Gly Tyr Arg Ph #e Phe Ala Asn Lys430                 4 #35                 4 #40Asp Ile Thr Gly Leu Thr Asn Ala Phe Ser Ar #g Ile Ser Ser Arg445                 4 #50                 4 #55Ser Gly Ser Ile Thr Gln Gln Ala Ile Gln Le #u Glu Ser Lys Ala460                 4 #65                 4 #70Leu Lys Ile Thr Gly Arg Lys Arg Val Asn Gl #y Thr Val Pro Val475                 4 #80                 4 #85Asp Ser Thr Val Gly Asn Asp Thr Phe Phe Va #l Val Thr Trp Thr490                 4 #95                 5 #00Ile Gln Lys Pro Glu Ile Val Leu Gln Asp Pr #o Lys Gly Lys Lys505                 5 #10                 5 #15Tyr Lys Thr Ser Asp Phe Lys Glu Asp Lys Le #u Asn Ile Arg Ser520                 5 #25                 5 #30Ala Arg Leu Gln Ile Pro Gly Ile Ala Glu Th #r Gly Thr Trp Thr535                 5 #40                 5 #45Tyr Ser Leu Leu Asn Asn His Ala Ser Ser Gl #n Met Leu Thr Val550                 5 #55                 5 #60Thr Val Thr Thr Arg Ala Arg Ser Pro Thr Il #e Pro Pro Val Ile565                 5 #70                 5 #75Ala Thr Ala His Met Ser Gln His Thr Ala Hi #s Tyr Pro Ser Pro580                 5 #85                 5 #90Met Ile Val Tyr Ala Gln Val Ser Gln Gly Ph #e Leu Pro Val Leu595                 6 #00                 6 #05Gly Ile Ser Val Ile Ala Ile Ile Glu Thr Gl #u Asp Gly His Gln610                 6 #15                 6 #20Val Thr Leu Glu Leu Trp Asp Asn Gly Ala Gl #y Arg Asp Thr Val625                 6 #30                 6 #35Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe Th #r Asp Tyr Tyr Gly640                 6 #45                 6 #50Asn Gly Arg Tyr Ser Leu Lys Val His Ala Gl #n Ala Arg Asn Asn655                 6 #60                 6 #65Thr Ala Arg Leu Asn Leu Arg Gln Pro Gln As #n Lys Val Leu Tyr670                 6 #75                 6 #80Val Pro Gly Tyr Val Glu Asn Gly Lys Ile Il #e Leu Asn Pro Pro685                 6 #90                 6 #95Arg Pro Glu Val Lys Asp Asp Leu Ala Lys Al #a Lys Ile Glu Asp700                 7 #05                 7 #10Phe Ser Arg Leu Thr Ser Gly Gly Ser Phe Th #r Val Ser Gly Ala715                 7 #20                 7 #25Pro Pro Pro Gly Asn His Pro Ser Val Phe Pr #o Pro Ser Lys Ile730                 7 #35                 7 #40Thr Asp Leu Glu Ala Lys Phe Lys Glu Asp Ty #r Ile Gln Leu Ser745                 7 #50                 7 #55Trp Thr Ala Pro Gly Asn Val Leu Asp Lys Gl #y Lys Ala Glu Ser760                 7 #65                 7 #70             774<210> SEQ ID NO 12 <211> LENGTH: 821 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Variant of Lu-ECAM-1 from # bovine endothelial      cells <400> SEQUENCE: 12Met Val Leu Cys Leu Asn Val Ile Leu Phe Le #u Thr Leu His Leu    -20               #   -15               #   -10Leu Pro Gly Met Lys Ser Ser Met Val Asn Le #u Ile Asn Asn Gly    -5               #     1              #  5Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp10                   # 15                  # 20Glu Lys Leu Ile Glu Asn Ile Lys Glu Met Va #l Thr Glu Ala Ser25                   # 30                  # 35Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Va #l Tyr Phe Arg Asn40                   # 45                  # 50Val Ser Ile Leu Ile Pro Met Thr Trp Lys Se #r Lys Ser Glu Tyr55                   # 60                  # 65Phe Ile Pro Lys Gln Glu Ser Tyr Asp Gln Al #a Asp Val Ile Val70                   # 75                  # 80Ala Asn Pro Tyr Leu Lys Tyr Gly Asp Asp Pr #o Tyr Thr Leu Gln85                   # 90                  # 95Tyr Gly Arg Cys Gly Glu Lys Gly Lys Tyr Il #e His Phe Thr Pro100                 1 #05                 1 #10Asn Phe Leu Leu Thr Asn Asn Phe His Ile Ty #r Gly Ser Arg Gly115                 1 #20                 1 #25Arg Val Phe Val His Glu Trp Ala His Leu Ar #g Trp Gly Ile Phe130                 1 #35                 1 #40Asp Glu Tyr Asn Val Asp Gln Pro Phe Tyr Il #e Ser Arg Lys Asn145                 1 #50                 1 #55Thr Ile Glu Ala Thr Arg Cys Ser Thr His Il #e Thr Gly Ile Asn160                 1 #65                 1 #70Val Val Phe Lys Lys Cys Pro Gly Gly Ser Cy #s Ile Thr Ser Leu175                 1 #80                 1 #85Cys Arg Arg Asp Ser Gln Thr Gly Leu Tyr Gl #u Ala Lys Cys Thr190                 1 #95                 2 #00Phe Leu Pro Lys Lys Ser Gln Thr Ala Lys Gl #u Ser Ile Met Phe205                 2 #10                 2 #15Met Pro Ser Leu His Ser Val Thr Glu Phe Cy #s Thr Glu Lys Thr220                 2 #25                 2 #30His Asn Thr Glu Ala Pro Asn Leu Gln Asn Ly #s Met Cys Asn Gly235                 2 #40                 2 #45Lys Ser Thr Trp Asp Val Ile Met Asn Ser Va #l Asp Phe Gln Asn250                 2 #55                 2 #60Thr Ser Pro Met Thr Glu Met Asn Pro Pro Th #r His Pro Thr Phe265                 2 #70                 2 #75Ser Leu Leu Lys Ser Lys Gln Arg Val Val Cy #s Leu Val Leu Asp280                 2 #85                 2 #90Lys Ser Gly Ser Met Ser Ala Glu Asp Arg Le #u Phe Gln Met Asn295                 3 #00                 3 #05Gln Ala Ala Glu Leu Tyr Leu Ile Gln Val Il #e Glu Lys Gly Ser310                 3 #15                 3 #20Leu Val Gly Met Val Thr Phe Asp Ser Val Al #a Glu Ile Gln Asn325                 3 #30                 3 #35His Leu Thr Arg Ile Thr Asp Asp Asn Val Ty #r Gln Lys Ile Thr340                 3 #45                 3 #50Ala Lys Leu Pro Gln Val Ala Asn Gly Gly Th #r Ser Ile Cys Arg355                 3 #60                 3 #65Gly Leu Lys Ala Gly Phe Gln Ala Ile Ile Hi #s Ser Asp Gln Ser370                 3 #75                 3 #80Thr Ser Gly Ser Glu Ile Ile Leu Leu Thr As #p Gly Glu Asp Asn385                 3 #90                 3 #95Glu Ile Asn Ser Cys Phe Glu Asp Val Lys Ar #g Ser Gly Ala Ile400                 4 #05                 4 #10Ile His Thr Ile Ala Leu Gly Pro Ser Ala Al #a Lys Glu Leu Glu415                 4 #20                 4 #25Thr Lys Ser Asn Met Thr Gly Gly Tyr Arg Ph #e Phe Ala Asn Lys430                 4 #35                 4 #40Asp Ile Thr Gly Leu Thr Asn Ala Phe Ser Ar #g Ile Ser Ser Arg445                 4 #50                 4 #55Ser Gly Ser Ile Thr Gln Gln Ala Ile Gln Le #u Glu Ser Lys Ala460                 4 #65                 4 #70Leu Lys Ile Thr Gly Arg Lys Arg Val Asn Gl #y Thr Val Pro Val475                 4 #80                 4 #85Asp Ser Thr Val Gly Asn Asp Thr Phe Phe Va #l Val Thr Trp Thr490                 4 #95                 5 #00Ile Gln Lys Pro Glu Ile Val Leu Gln Asp Pr #o Lys Gly Lys Lys505                 5 #10                 5 #15Tyr Lys Thr Ser Asp Phe Lys Glu Asp Lys Le #u Asn Ile Arg Ser520                 5 #25                 5 #30Ala Arg Leu Gln Ile Pro Gly Ile Ala Glu Th #r Gly Thr Trp Thr535                 5 #40                 5 #45Tyr Ser Leu Leu Asn Asn His Ala Ser Ser Gl #n Met Leu Thr Val550                 5 #55                 5 #60Thr Val Thr Thr Arg Ala Arg Ser Pro Thr Il #e Pro Pro Val Ile565                 5 #70                 5 #75Ala Thr Ala His Met Ser Gln His Thr Ala Hi #s Tyr Pro Ser Pro580                 5 #85                 5 #90Met Ile Val Tyr Ala Gln Val Ser Gln Gly Ph #e Leu Pro Val Leu595                 6 #00                 6 #05Gly Ile Ser Val Ile Ala Ile Ile Glu Thr Gl #u Asp Gly His Gln610                 6 #15                 6 #20Val Thr Leu Glu Leu Trp Asp Asn Gly Ala Gl #y Arg Asp Thr Val625                 6 #30                 6 #35Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe Th #r Asp Tyr Tyr Gly640                 6 #45                 6 #50Asn Gly Arg Tyr Ser Leu Lys Val His Ala Gl #n Ala Arg Asn Asn655                 6 #60                 6 #65Thr Ala Arg Leu Asn Leu Arg Gln Pro Gln As #n Lys Val Leu Tyr670                 6 #75                 6 #80Val Pro Gly Tyr Val Glu Asn Gly Lys Ile Il #e Leu Asn Pro Pro685                 6 #90                 6 #95Arg Pro Glu Val Lys Asp Asp Leu Ala Lys Al #a Lys Ile Glu Asp700                 7 #05                 7 #10Phe Ser Arg Leu Thr Ser Gly Gly Ser Phe Th #r Val Ser Gly Ala715                 7 #20                 7 #25Pro Pro Pro Gly Asn His Pro Ser Val Phe Pr #o Pro Ser Lys Ile730                 7 #35                 7 #40Thr Asp Leu Glu Ala Lys Phe Lys Glu Asp Ty #r Ile Gln Leu Ser745                 7 #50                 7 #55Trp Thr Ala Pro Gly Asn Val Leu Asp Lys Gl #y Lys Ala Ala Ser760                 7 #65                 7 #70Gly Ser Phe Pro Met Ser Arg Phe Ser His Gl #n Val Ala Lys Val775                 7 #80                 7 #85Leu Glu Leu Gln Leu Gln His Gln Ser Phe Gl #n 790                 7#95                 8 #00 <210> SEQ ID NO 13 <211> LENGTH: 342<212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Variant of Lu-ECAM-1 from # bovine endothelial      cells <400> SEQUENCE: 13Met Val Leu Cys Leu Asn Val Ile Leu Phe Le #u Thr Leu His Leu    -20               #   -15               #   -10Leu Pro Gly Met Lys Ser Ser Met Val Asn Le #u Ile Asn Asn Gly     -5              #     1              #  5Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp10                   # 15                  # 20Glu Lys Leu Ile Glu Asn Ile Lys Glu Met Va #l Thr Glu Ala Ser25                   # 30                  # 35Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Va #l Tyr Phe Arg Asn40                   # 45                  # 50Val Ser Ile Leu Ile Pro Met Thr Trp Lys Se #r Lys Ser Glu Tyr55                   # 60                  # 65Phe Ile Pro Lys Gln Glu Ser Tyr Asp Gln Al #a Asp Val Ile Val 70                  # 75                  # 80Ala Asn Pro Tyr Leu Lys Tyr Gly Asp Asp Pr #o Tyr Thr Leu Gln85                   # 90                  # 95Tyr Gly Arg Cys Gly Glu Lys Gly Lys Tyr Il #e His Phe Thr Pro100                 1 #05                 1 #10Asn Phe Leu Leu Thr Asn Asn Phe His Ile Ty #r Gly Ser Arg Gly115                 1 #20                 1 #25Arg Val Phe Val His Glu Trp Ala His Leu Ar #g Trp Gly Ile Phe130                 1 #35                 1 #40Asp Glu Tyr Asn Val Asp Gln Pro Phe Tyr Il #e Ser Arg Lys Asn145                 1 #50                 1 #55Thr Ile Glu Ala Thr Arg Cys Ser Thr His Il #e Thr Gly Ile Asn160                 1 #65                 1 #70Val Val Phe Lys Lys Cys Pro Gly Gly Ser Cy #s Ile Thr Ser Leu175                 1 #80                 1 #85Cys Arg Arg Asp Ser Gln Thr Gly Leu Tyr Gl #u Ala Lys Cys Thr190                 1 #95                 2 #00Phe Leu Pro Lys Lys Ser Gln Thr Ala Lys Gl #u Ser Ile Met Phe205                 2 #10                 2 #15Met Pro Ser Leu His Ser Val Thr Glu Phe Cy #s Thr Glu Lys Thr220                 2 #25                 2 #30His Asn Thr Glu Ala Pro Asn Leu Gln Asn Ly #s Met Cys Asn Gly235                 2 #40                 2 #45Lys Ser Thr Trp Asp Val Ile Met Asn Ser Va #l Asp Phe Gln Asn250                 2 #55                 2 #60Thr Ser Pro Met Thr Glu Met Asn Pro Pro Th #r His Pro Thr Phe265                 2 #70                 2 #75Ser Leu Leu Lys Ser Lys Gln Arg Val Val Cy #s Leu Val Leu Asp280                 2 #85                 2 #90Lys Ser Gly Ser Met Ser Ala Glu Asp Ile Ty #r Leu Leu Ala Leu295                 3 #00                 3 #05Leu Ile Lys Ile Phe Lys Leu Ile Gly Asn Th #r Ile 310                 3#15                 3 #20 321 <210> SEQ ID NO 14 <211> LENGTH: 335<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide probe <400> SEQUENCE: 14caacagctac attataagaa taagtaagag tttcatggat cgtcaagaag  #              50attttgacaa tgcgacttta gtgaatactt ctaatctaat acctaaggag  #             100gccggatcaa aagaaaattt tgaatttaag ccagaacatt ttagagtaga  #             150aaatggcacc aaattctata tttcagtcca agccatcaac gaagccaatc  #             200tcatctcaga ggtttctcac attgtacaag caatcaaatt tattcctcta  #             250ccagaagaca gtgtccatga tctgggtacc aagatttctg aaatcactct  #             300 ggcaatttta ggattaccaa tgattttctc tgtat       #                   #      335 <210> SEQ ID NO 15 <211> LENGTH: 17<212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide <400> SEQUENCE: 15Glu Asp Glu Lys Leu Ile Glu Asn Ile Lys Gl #u Met Val Thr Glu                 5  #                   #10                   #15Ala Ser      17 <210> SEQ ID NO 16 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic peptide <400> SEQUENCE: 16Gln Asp Pro Lys Gly Lys Lys Tyr Lys Thr Se #r Asp Phe Lys Glu 1               5   #                 10  #                 15 Asp Lys     17 <210> SEQ ID NO 17 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 17atgttcaact catattactg gtat           #                  #                24 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 18tgtaggtttg gagcttctgt             #                  #                   # 20 <210> SEQ ID NO 19 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 19cacagacagg gctgtatgaa             #                  #                   # 20 <210> SEQ ID NO 20 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification Primer <400> SEQUENCE: 20ggagatgtat tctgaaagtc aac            #                  #                23 <210> SEQ ID NO 21 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 21atgttcaact catattactg gtac           #                  #                24 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 22tgtaggtttg gagcttccac             #                  #                   # 20 <210> SEQ ID NO 23 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 23cacagacagg gctgtatgag             #                  #                   # 20 <210> SEQ ID NO 24 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 24ggagatgtat tttgaaagtc agt            #                  #                23 <210> SEQ ID NO 25 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 25actgaattca gcagactaac ctctggaggg tc        #                  #          32 <210> SEQ ID NO 26 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 26tctactagta gctttagcta ctgaagaaca ag        #                  #          32 <210> SEQ ID NO 27 <211> LENGTH: 3007 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 27taacccgcat tttccaaaga gaggaatcac agggagatgt acagca atg #ggg            52 cca ttt aag agt tct gtg ttc atc ttg att ct#t cac ctt cta gaa            #97ggg gcc ctg agt aat tca ctc att cag ctg aa#c aac aat ggc tat          14 #2gaa ggc att gtc gtt gca atc gac ccc aat gt#g cca gaa gat gaa          18 #7aca ctc att caa caa ata aag gac atg gtg ac#c cag gca tct ctg          23 #2tat ctg ttt gaa gct aca gga aag cga ttt ta#t ttc aaa aat gtt          27 #7gcc att ttg att cct gaa aca tgg aag aca aa#g gct gac tat gtg          32 #2aga cca aaa ctt gag acc tac aaa aat gct ga#t gtt ctg gtt gct          36 #7gag tct act cct cca ggt aat gat gaa ccc ta#c act gag cag atg          41 #2ggc aac tgt gga gag aag ggt gaa agg atc ca#c ctc act cct gat          45 #7ttc att gca gga aaa aag tta gct gaa tat gg#a cca caa ggt aag          50 #2gca ttt gtc cat gag tgg gct cat cta cga tg#g gga gta ttt gac          54 #7gag tac aat aat gat gag aaa ttc tac tta tc#c aat gga aga ata          59 #2caa gca gta aga tgt tca gca ggt att act gg#t aca aat gta gta          63 #7aag aag tgt cag gga ggc agc tgt tac acc aa#a aga tgc aca ttc          68 #2aat aaa gtt aca gga ctc tat gaa aaa gga tg#t gag ttt gtt ctc          72 #7caa tcc cgc cag acg gag aag gct tct ata at#g ttt gca caa cat          77 #2gtt gat tct ata gtt gaa ttc tgt aca gaa ca#a aac cac aac aaa          81 #7gaa gct cca aac aag caa aat caa aaa tgc aa#t ctc cga agc aca          86 #2tgg gaa gtg atc cgt gat tct gag gac ttt aa#g aaa acc act cct          90 #7atg aca aca cag cca cca aat ccc acc ttc tc#a ttg ctg cag att          95 #2gga caa aga att gtg tgt tta gtc ctt gac aa#a tct gga agc atg          99 #7gcg act ggt aac cgc ctc aat cga ctg aat ca#a gca ggc cag ctt         1042ttc ctg ctg cag aca gtt gag ctg ggg tcc tg#g gtt ggg atg gtg         1087aca ttt gac agt gct gcc cat gta caa agt ga#a ctc ata cag ata         1132aac agt ggc agt gac agg gac aca ctc gcc aa#a aga tta cct gca         1177gca gct tca gga ggg acg tcc atc tgc agc gg#g ctt cga tcg gca         1222ttt act gtg att agg aag aaa tat cca act ga#t gga tct gaa att         1267gtg ctg ctg acg gat ggg gaa gac aac act at#a agt ggg tgc ttt         1312aac gag gtc aaa caa agt ggt gcc atc atc ca#c aca gtc gct ttg         1357ggg ccc tct gca gct caa gaa cta gag gag ct#g tcc aaa atg aca         1402gga ggt tta cag aca tat gct tca gat caa gt#t cag aac aat ggc         1447ctc att gat gct ttt ggg gcc ctt tca tca gg#a aat gga gct gtc         1492tct cag cgc tcc atc cag ctt gag agt aag gg#a tta acc ctc cag         1537aac agc cag tgg atg aat ggc aca gtg atc gt#g gac agc acc gtg         1582gga aag gac act ttg ttt ctt atc acc tgg ac#a acg cag cct ccc         1627caa atc ctt ctc tgg gat ccc agt gga cag aa#g caa ggt ggc ttt         1672gta gtg gac aaa aac acc aaa atg gcc tac ct#c caa atc cca ggc         1717att gct aag gtt ggc act tgg aaa tac agt ct#g caa gca agc tca         1762caa acc ttg acc ctg act gtc acg tcc cgt gc#g tcc aat gct acc         1807ctg cct cca att aca gtg act tcc aaa acg aa#c aag gac acc agc         1852aaa ttc ccc agc cct ctg gta gtt tat gca aa#t att cgc caa gga         1897gcc tcc cca att ctc agg gcc agt gtc aca gc#c ctg att gaa tca         1942gtg aat gga aaa aca gtt acc ttg gaa cta ct#g gat aat gga gca         1987ggt gct gat gct act aag gat gac ggt gtc ta#c tca agg tat ttc         2032aca act tat gac acg aat ggt aga tac agt gt#a aaa gtg cgg gct         2077ctg gga gga gtt aac gca gcc aga cgg aga gt#g ata ccc cag cag         2122agt gga gca ctg tac ata cct ggc tgg att ga#g aat gat gaa ata         2167caa tgg aat cca cca aga cct gaa att aat aa#g gat gat gtt caa         2212cac aag caa gtg tgt ttc agc aga aca tcc tc#g gga ggc tca ttt         2257gtg gct tct gat gtc cca aat gct ccc ata cc#t gat ctc ttc cca         2302cct ggc caa atc acc gac ctg aag gcg gaa at#t cac ggg ggc agt         2347ctc att aat ctg act tgg aca gct cct ggg ga#t gat tat gac cat         2392gga aca gct cac aag tat atc att cga ata ag#t aca agt att ctt         2437gat ctc aga gac aag ttc aat gaa tct ctt ca#a gtg aat act act         2482gct ctc atc cca aag gaa gcc aac tct gag ga#a gtc ttt ttg ttt         2527aaa cca gaa aac att act ttt gaa aat ggc ac#a gat ctt ttc att         2572gct att cag gct gtt gat aag gtc gat ctg aa#a tca gaa ata tcc         2617aac att gca cga gta tct ttg ttt att cct cc#a cag act ccg cca         2662gag aca cct agt cct gat gaa acg tct gct cc#t tgt cct aat att         2707cat atc aac agc acc att cct ggc att cac at#t tta aaa att atg         2752tgg aag tgg ata gga gaa ctg cag ctg tca at#a gcc tagggctgaa          2798tttttgtcag ataaataaaa taaatcattc atcctttttt ttgattataa  #            2848aattttttaa aatgtatttt agaattcctg tagggggcga tatactaaat  #            2898gtatatagta catttatact aaatgtattc ctgtaggggg cgatatacta  #            2948aatgtatttt agaattcctg tagggggcga taaaataaaa tgctaaacaa  #            2998 ctggggaaa                 #                  #                   #       3007 <210> SEQ ID NO 28 <211> LENGTH: 914<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 28Met Gly Pro Phe Lys Ser Ser Val Phe Ile Le #u Ile Leu His Leu1                5   #                 10  #                 15Leu Glu Gly Ala Leu Ser Asn Ser Leu Ile Gl #n Leu Asn Asn Asn                 20  #                 25  #                 30Gly Tyr Glu Gly Ile Val Val Ala Ile Asp Pr #o Asn Val Pro Glu                 35  #                 40  #                 45Asp Glu Thr Leu Ile Gln Gln Ile Lys Asp Me #t Val Thr Gln Ala                 50  #                 55  #                 60Ser Leu Tyr Leu Phe Glu Ala Thr Gly Lys Ar #g Phe Tyr Phe Lys                 65  #                 70  #                 75Asn Val Ala Ile Leu Ile Pro Glu Thr Trp Ly #s Thr Lys Ala Asp                 80  #                 85  #                 90Tyr Val Arg Pro Lys Leu Glu Thr Tyr Lys As #n Ala Asp Val Leu                 95  #                100  #                105Val Ala Glu Ser Thr Pro Pro Gly Asn Asp Gl #u Pro Tyr Thr Glu                110   #               115   #               120Gln Met Gly Asn Cys Gly Glu Lys Gly Glu Ar #g Ile His Leu Thr                125   #               130   #               135Pro Asp Phe Ile Ala Gly Lys Lys Leu Ala Gl #u Tyr Gly Pro Gln                140   #               145   #               150Gly Lys Ala Phe Val His Glu Trp Ala His Le #u Arg Trp Gly Val                155   #               160   #               165Phe Asp Glu Tyr Asn Asn Asp Glu Lys Phe Ty #r Leu Ser Asn Gly                170   #               175   #               180Arg Ile Gln Ala Val Arg Cys Ser Ala Gly Il #e Thr Gly Thr Asn                185   #               190   #               195Val Val Lys Lys Cys Gln Gly Gly Ser Cys Ty #r Thr Lys Arg Cys                200   #               205   #               210Thr Phe Asn Lys Val Thr Gly Leu Tyr Glu Ly #s Gly Cys Glu Phe                215   #               220   #               225Val Leu Gln Ser Arg Gln Thr Glu Lys Ala Se #r Ile Met Phe Ala                230   #               235   #               240Gln His Val Asp Ser Ile Val Glu Phe Cys Th #r Glu Gln Asn His                245   #               250   #               255Asn Lys Glu Ala Pro Asn Lys Gln Asn Gln Ly #s Cys Asn Leu Arg                260   #               265   #               270Ser Thr Trp Glu Val Ile Arg Asp Ser Glu As #p Phe Lys Lys Thr                275   #               280   #               285Thr Pro Met Thr Thr Gln Pro Pro Asn Pro Th #r Phe Ser Leu Leu                290   #               295   #               300Gln Ile Gly Gln Arg Ile Val Cys Leu Val Le #u Asp Lys Ser Gly                305   #               310   #               315Ser Met Ala Thr Gly Asn Arg Leu Asn Arg Le #u Asn Gln Ala Gly                320   #               325   #               330Gln Leu Phe Leu Leu Gln Thr Val Glu Leu Gl #y Ser Trp Val Gly                335   #               340   #               345Met Val Thr Phe Asp Ser Ala Ala His Val Gl #n Ser Glu Leu Ile                350   #               355   #               360Gln Ile Asn Ser Gly Ser Asp Arg Asp Thr Le #u Ala Lys Arg Leu                365   #               370   #               375Pro Ala Ala Ala Ser Gly Gly Thr Ser Ile Cy #s Ser Gly Leu Arg                380   #               385   #               390Ser Ala Phe Thr Val Ile Arg Lys Lys Tyr Pr #o Thr Asp Gly Ser                395   #               400   #               405Glu Ile Val Leu Leu Thr Asp Gly Glu Asp As #n Thr Ile Ser Gly                410   #               415   #               420Cys Phe Asn Glu Val Lys Gln Ser Gly Ala Il #e Ile His Thr Val                425   #               430   #               435Ala Leu Gly Pro Ser Ala Ala Gln Glu Leu Gl #u Glu Leu Ser Lys                440   #               445   #               450Met Thr Gly Gly Leu Gln Thr Tyr Ala Ser As #p Gln Val Gln Asn                455   #               460   #               465Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu Se #r Ser Gly Asn Gly                470   #               475   #               480Ala Val Ser Gln Arg Ser Ile Gln Leu Glu Se #r Lys Gly Leu Thr                485   #               490   #               495Leu Gln Asn Ser Gln Trp Met Asn Gly Thr Va #l Ile Val Asp Ser                500   #               505   #               510Thr Val Gly Lys Asp Thr Leu Phe Leu Ile Th #r Trp Thr Thr Gln                515   #               520   #               525Pro Pro Gln Ile Leu Leu Trp Asp Pro Ser Gl #y Gln Lys Gln Gly                530   #               535   #               540Gly Phe Val Val Asp Lys Asn Thr Lys Met Al #a Tyr Leu Gln Ile                545   #               550   #               555Pro Gly Ile Ala Lys Val Gly Thr Trp Lys Ty #r Ser Leu Gln Ala                560   #               565   #               570Ser Ser Gln Thr Leu Thr Leu Thr Val Thr Se #r Arg Ala Ser Asn                575   #               580   #               585Ala Thr Leu Pro Pro Ile Thr Val Thr Ser Ly #s Thr Asn Lys Asp                590   #               595   #               600Thr Ser Lys Phe Pro Ser Pro Leu Val Val Ty #r Ala Asn Ile Arg                605   #               610   #               615Gln Gly Ala Ser Pro Ile Leu Arg Ala Ser Va #l Thr Ala Leu Ile                620   #               625   #               630Glu Ser Val Asn Gly Lys Thr Val Thr Leu Gl #n Leu Leu Asp Asn                635   #               640   #               645Gly Ala Gly Ala Asp Ala Thr Lys Asp Asp Gl #y Val Tyr Ser Arg                650   #               655   #               660Tyr Phe Thr Thr Tyr Asp Thr Asn Gly Arg Ty #r Ser Val Lys Val                665   #               670   #               675Arg Ala Leu Gly Gly Val Asn Ala Ala Arg Ar #g Arg Val Ile Pro                680   #               685   #               690Gln Gln Ser Gly Ala Leu Tyr Ile Pro Gly Tr #p Ile Glu Asn Asp                695   #               700   #               705Glu Ile Gln Trp Asn Pro Pro Arg Pro Glu Il #e Asn Lys Asp Asp                710   #               715   #               720Val Gln His Lys Gln Val Cys Phe Ser Arg Th #r Ser Ser Gly Gly                725   #               730   #               735Ser Phe Val Ala Ser Asp Val Pro Asn Ala Pr #o Ile Pro Asp Leu                740   #               745   #               750Phe Pro Pro Gly Gln Ile Thr Asp Leu Lys Al #a Glu Ile His Gly                755   #               760   #               765Gly Ser Leu Ile Asn Leu Thr Trp Thr Ala Pr #o Gly Asp Asp Tyr                770   #               775   #               780Asp His Gly Thr Ala His Lys Tyr Ile Ile Ar #g Ile Ser Thr Ser                785   #               790   #               795Ile Leu Asp Leu Arg Asp Lys Phe Asn Glu Se #r Leu Gln Val Asn                800   #               805   #               810Thr Thr Ala Leu Ile Pro Lys Glu Ala Asn Se #r Glu Glu Val Phe                815   #               820   #               825Leu Phe Lys Pro Glu Asn Ile Thr Phe Glu As #n Gly Thr Asp Leu                830   #               835   #               840Phe Ile Ala Ile Gln Ala Val Asp Lys Val As #p Leu Lys Ser Glu                845   #               850   #               855Ile Ser Asn Ile Ala Arg Val Ser Leu Phe Il #e Pro Pro Gln Thr                860   #               865   #               870Pro Pro Glu Thr Pro Ser Pro Asp Glu Thr Se #r Ala Pro Cys Pro                875   #               880   #               885Asn Ile His Ile Asn Ser Thr Ile Pro Gly Il #e His Ile Leu Lys                890   #               895   #               900Ile Met Trp Lys Trp Ile Gly Glu Leu Gln Le #u Ser Ile Ala                905   #               910   #           914<210> SEQ ID NO 29 <211> LENGTH: 3418 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 29tttgtttaac atgcaaga atg gtg ttc agt ctg aag gtg #att ctc ttc           48 cta tcc ttg ctt ctc tcg cct gta ttg aaa ag#c tca ctg gta act            #93ttg aat aac aat gga tat gat ggc att gtg at#t gca att aat ccc          13 #8agt gta cca gaa gat gaa aaa ctc att caa aa#c ata aag gaa atg          18 #3gta act gaa gca tct act cac ctg ttt cat gc#c acc aaa caa aga          22 #8gct tat ttc agg aat gta agc att tta att cc#a atg acc tac aaa          27 #3tca aaa tct gag tac tta atc cca aaa caa ga#a aca tat gac cag          31 #8gca gat gtc ata gtt gct gat ctt tac ctg aa#a tac gga gat gat          36 #3ccc tat aca ctt caa tat gga caa tgt gga ga#t aaa gga caa tat          40 #8ata cat ttt act cca aac ttc ttg ttg act aa#t aac ttg gct acc          45 #3tat ggg cct cga ggt aaa gta ttt gtc cat gg#g tgg gcc cat ctc          49 #8cgg tgg gga gta ttt gat gag tat aat gtg ga#c cag cca ttc tat          54 #3att tcc aga aga aac act act gaa gca aca ag#a tgt tcc act cgt          58 #8att act gtt tac atg gtt ttg aac gaa tgc aa#g ggg gcc agc tgt          63 #3ata gca cga cca ttc aga cgt gac tca cag ac#a ggg ctg tat gaa          67 #8gca aaa tgt aca ttt atc cca aag aga tcc ca#g act gcc aag gaa          72 #3tcc att gtg ttt atg caa aat ctt gat tct gt#g act gaa ttt tgt          76 #8act gaa aaa aca cac aat aaa gaa gct cca aa#c cta tat aac aaa          81 #3atg tgc aat cac aga agc aca tgg gat gta at#c atg agc tct gaa          85 #8gat ttt cag cat tta tct ccc atg aca gaa at#a aat tta cct cgt          90 #3cct aca ttt tca ttg ctc aag tcc aaa cag cg#t gta gtc tgt ttg          94 #8gta ctt gat aaa tct gga agc atg aat gca ga#a gac cgt ctc ttt          99 #3cga atg aat caa gca gca gaa ttg tac ttg at#t caa att att gaa         1038aag gga tcc ttg gtt ggg ttg gtc aca ttt ga#c agt ttt gct aaa         1083atc caa agt aag ctc ata aaa ata att gat ga#t aac act tac caa         1128aag atc act gca aac ctg cct caa gaa gct ga#t ggt ggc act tca         1173att tgc agg gga ctc aaa gca gga ttt cag gc#a att ccc cag agt         1218aat cag agt act ttc ggt tct gaa atc ata tt#a cta aca gat ggg         1263gaa gat tat caa ata agc tta tgc ttt gga ga#g gta aaa caa agt         1308ggc aca gtc atc cac acc att gct ctg ggg cc#g tct gct gac gaa         1353gaa ctg gag acc ctg tca aat atg aca gga tt#a cat aag gga cac         1398tgt tat act gaa agt tca tat agt gct ggg aa#g ttc atc ttt tgt         1443gga cat cgt ttt tat gcc cat aaa aac ata aa#t ggc ctt att gat         1488gct ttc agc aga att tca tct aga agt ggc ag#c atc tct cag cag         1533gct ctt cag ttg gaa agt aaa act ttg aat at#c cca gcg aag aaa         1578tgg ata aat ggt aca gtg cct gtg gat agt ac#a gtt aga aat gat         1623act tcc ttt gtt gtc aca tgg acg ata caa aa#g cca gca ata att         1668ctt caa gat cca aaa gga aaa aaa tat act ac#c tca gat ttt caa         1713gaa ggt gaa cta aat att cgg tct gcc cgt ct#t cga ata cca ggt         1758att gca gag aca ggc act tgg act tac agc gt#t cga aac aat cat         1803acc aaa tct caa ttg cta act gtg aca atg ac#c act cga gca aga         1848agc cct acc aca ctc cca gta att gca act gc#t cac atg agt caa         1893aat aca gct cat tac cct agc cca gtg att gt#t tat gca tgt gtc         1938agt caa ggg ttt ctt cct gtt ctg gga atc aa#t gta aca gcc att         1983ata gaa aat gaa gag gga cat caa gta aca tt#g gag ctc tgc gac         2028aat ggc gca ggt gct gat tct gtc aag aat ga#t ggc atc tac tca         2073agg tat ttt aca gat tac cat gga aat ggt ag#a tac agt tta aaa         2118gtg ctt acc cag gca aga aaa aac aca gct ag#g cta agt caa caa         2163cag aat aaa gct ctg tat gta ccg cgc tat gc#t gaa aat gga aaa         2208att ata ctg aac cca tcc aaa cct gaa gtc ac#a gat gat gtg gaa         2253gga gct caa aca gac gac ttc agc aga ctc ac#c tct gga ggg tcg         2298ttt act gta tca gga gtg cct cct aat ggt aa#t cat tct cag gtg         2343ttc tca cct ggt aaa att gta gac ctc gag gc#t aag ttt caa gga         2388gat cat att caa ctt tca tgg act gcc cct gg#c aag gtc ctc gat         2433aaa gga aga gct gag agc tac att ata aga at#a agt aaa cat ttc         2478ctg gac ctc caa gaa gat ttt gat aaa gct gc#t tta ata aat act         2523tct ggt ctg ata cct aag gag cct ggt tca gt#a gaa agt ttt gaa         2568ttt aaa cca gaa cct tct aaa ata gag aat gg#t acg aca ttc tat         2613att gca att caa gcc atc cat gaa gcc aat gt#c acc tca gag gtt         2658tca aac att gca caa gca act aac ttt att cc#t cca cag gaa ccc         2703agc att cct gat ctg ggt acc aat att tct gc#a atc agt ttg gca         2748att ttt gga tta gct gta att tta tct ata tt#t tat act aga aat         2793tat att aga act caa att caa tgt tat aca ta#c ttg gta aac att         2838tat tta aaa ttt aat tta cta tac tta ttg tc#t att ata aag ctc         2883att ata ata tat aaa gtg aag tac aaa agt tg#t aag ttt cct aat         2928tac ttg att aat tat tac tat ttg agt tat ta#t atg tta atc aaa         2973atg agt ata tca ttt cct gtc tgg aat aat cc#a ctc att aat ttt         3018taatatgaaa agatatatat ttgtacttgt aagcatttta agaaacattt  #            3068ttaaagtgtg ctacaaattc atttggtgta ctaacatcaa aatgtatcca  #            3118agccatttaa aaaatattta tatatacata gtagcaaata gttttataga  #            3168tttatttgta tcgcattttt tattacaaat gaatatttca tgtttatata  #            3218agctgtaatc aaaaaggact agtagtagta gtaaggaagt caaatttgtt  #            3268tttttatcat tgattataag tggtatattt gttttttgtc attgattaaa  #            3318agtgatttta gccctaggcc cgaaatgact agcaaatatc attttctgta  #            3368tgaattgtgg aacatcacaa taaaattatt tctgtgctga tgctaaaaaa  #            3418 <210> SEQ ID NO 30 <211> LENGTH: 1000 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 30Met Val Phe Ser Leu Lys Val Ile Leu Phe Le #u Ser Leu Leu Leu1                5   #                 10  #                 15Ser Pro Val Leu Lys Ser Ser Leu Val Thr Le #u Asn Asn Asn Gly                 20  #                 25  #                 30Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp                 35  #                 40  #                 45Glu Lys Leu Ile Gln Asn Ile Lys Glu Met Va #l Thr Gln Ala Ser                 50  #                 55  #                 60Thr His Leu Phe His Ala Thr Lys Gln Arg Al #a Tyr Phe Arg Asn                 65  #                 70  #                 75Val Ser Ile Leu Ile Pro Met Thr Tyr Lys Se #r Lys Ser Glu Tyr                 80  #                 85  #                 90Leu Ile Pro Lys Gln Glu Thr Tyr Asp Gln Al #a Asp Val Ile Val                 95  #                100  #                105Ala Asp Leu Tyr Leu Lys Tyr Gly Asp Asp Pr #o Tyr Thr Leu Gln                110   #               115   #               120Tyr Gly Gln Cys Gly Asp Lys Gly Gln Tyr Il #e His Phe Thr Pro                125   #               130   #               135Asn Phe Leu Leu Thr Asn Asn Leu Ala Thr Ty #r Gly Pro Arg Gly                140   #               145   #               150Lys Val Phe Val His Gly Trp Ala His Leu Ar #g Trp Gly Val Phe                155   #               160   #               165Asp Glu Tyr Asn Val Asp Gln Pro Phe Tyr Il #e Ser Arg Arg Asn                170   #               175   #               180Thr Thr Glu Ala Thr Arg Cys Ser Thr Arg Il #e Thr Val Tyr Met                185   #               190   #               195Val Leu Asn Glu Cys Lys Gly Ala Ser Cys Il #e Ala Arg Pro Phe                200   #               205   #               210Arg Arg Asp Ser Gln Thr Gly Leu Tyr Glu Al #a Lys Cys Thr Phe                215   #               220   #               225Ile Pro Lys Arg Ser Gln Thr Ala Lys Glu Se #r Ile Val Phe Met                230   #               235   #               240Gln Asn Leu Asp Ser Val Thr Glu Phe Cys Th #r Glu Lys Thr His                245   #               250   #               255Asn Lys Glu Ala Pro Asn Leu Tyr Asn Lys Me #t Cys Asn His Arg                260   #               265   #               270Ser Thr Trp Asp Val Ile Met Ser Ser Glu As #p Phe Gln His Leu                275   #               280   #               285Ser Pro Met Thr Glu Ile Asn Leu Pro Arg Pr #o Thr Phe Ser Leu                290   #               295   #               300Leu Lys Ser Lys Gln Arg Val Val Cys Leu Va #l Leu Asp Lys Ser                305   #               310   #               315Gly Ser Met Asn Ala Glu Asp Arg Leu Phe Ar #g Met Asn Gln Ala                320   #               325   #               330Ala Glu Leu Tyr Leu Ile Gln Ile Ile Glu Ly #s Gly Ser Leu Val                335   #               340   #               345Gly Leu Val Thr Phe Asp Ser Phe Ala Lys Il #e Gln Ser Lys Leu                350   #               355   #               360Ile Lys Ile Ile Asp Asp Asn Thr Tyr Gln Ly #s Ile Thr Ala Asn                365   #               370   #               375Leu Pro Gln Glu Ala Asp Gly Gly Thr Ser Il #e Cys Arg Gly Leu                380   #               385   #               390Lys Ala Gly Phe Gln Ala Ile Pro Gln Ser As #n Gln Ser Thr Phe                395   #               400   #               405Gly Ser Glu Ile Ile Leu Leu Thr Asp Gly Gl #u Asp Tyr Gln Ile                410   #               415   #               420Ser Leu Cys Phe Gly Glu Val Lys Gln Ser Gl #y Thr Val Ile His                425   #               430   #               435Thr Ile Ala Leu Gly Pro Ser Ala Asp Glu Gl #u Leu Glu Thr Leu                440   #               445   #               450Ser Asn Met Thr Gly Leu His Lys Gly His Cy #s Tyr Thr Glu Ser                455   #               460   #               465Ser Tyr Ser Ala Gly Lys Phe Ile Phe Cys Gl #y His Arg Phe Tyr                470   #               475   #               480Ala His Lys Asn Ile Asn Gly Leu Ile Asp Al #a Phe Ser Arg Ile                485   #               490   #               495Ser Ser Arg Ser Gly Ser Ile Ser Gln Gln Al #a Leu Gln Leu Glu                500   #               505   #               510Ser Lys Thr Leu Asn Ile Pro Ala Lys Lys Tr #p Ile Asn Gly Thr                515   #               520   #               525Val Pro Val Asp Ser Thr Val Arg Asn Asp Th #r Ser Phe Val Val                530   #               535   #               540Thr Trp Thr Ile Gln Lys Pro Ala Ile Ile Le #u Gln Asp Pro Lys                545   #               550   #               555Gly Lys Lys Tyr Thr Thr Ser Asp Phe Gln Gl #u Gly Glu Leu Asn                560   #               565   #               570Ile Arg Ser Ala Arg Leu Arg Ile Pro Gly Il #e Ala Glu Thr Gly                575   #               580   #               585Ile Trp Thr Tyr Ser Val Arg Asn Asn His Th #r Lys Ser Gln Leu                590   #               595   #               600Leu Thr Val Thr Met Thr Thr Arg Ala Arg Se #r Pro Thr Thr Leu                605   #               610   #               615Pro Val Ile Ala Thr Ala His Ser Met Gln As #n Thr Ala His Tyr                620   #               625   #               630Pro Ser Pro Val Ile Val Tyr Ala Cys Val Se #r Gln Gly Phe Leu                635   #               640   #               645Pro Val Leu Gly Ile Asn Val Thr Ala Ile Il #e Glu Asn Glu Glu                650   #               655   #               660Gly His Gln Val Thr Leu Glu Leu Cys Asp As #n Gly Ala Gly Ala                665   #               670   #               675Asp Ser Val Lys Asn Asp Gly Ile Tyr Ser Ar #g Tyr Phe Thr Asp                680   #               685   #               690Tyr His Gly Asn Gly Arg Tyr Ser Leu Lys Va #l Leu Thr Gln Ala                695   #               700   #               705Arg Lys Asn Thr Ala Arg Leu Ser Gln Gln Gl #n Asn Lys Ala Leu                710   #               715   #               720Tyr Val Pro Arg Tyr Ala Glu Asn Gly Lys Il #e Ile Leu Asn Pro                725   #               730   #               735Ser Lys Pro Glu Val Thr Asp Asp Val Glu Gl #y Ala Gln Thr Asp                740   #               745   #               750Asp Phe Ser Arg Leu Thr Ser Gly Gly Ser Ph #e Thr Val Ser Gly                755   #               760   #               765Val Pro Pro Asn Gly Asn His Ser Gln Val Ph #e Ser Pro Gly Lys                770   #               775   #               780Ile Val Asp Leu Glu Ala Lys Phe Gln Gly As #p His Ile Gln Leu                785   #               790   #               795Ser Trp Thr Ala Pro Gly Lys Val Leu Asp Ly #s Gly Arg Ala Glu                800   #               805   #               810Ser Tyr Ile Ile Arg Ile Ser Lys His Phe Le #u Asp Leu Gln Glu                815   #               820   #               825Asp Phe Asp Lys Ala Ala Leu Ile Asn Thr Se #r Gly Leu Ile Pro                830   #               835   #               840Lys Glu Pro Gly Ser Val Glu Ser Phe Glu Ph #e Lys Pro Glu Pro                845   #               850   #               855Ser Lys Ile Glu Asn Gly Thr Thr Phe Tyr Il #e Ala Ile Gln Ala                860   #               865   #               870Ile His Glu Ala Asn Val Thr Ser Glu Val Se #r Asn Ile Ala Gln                875   #               880   #               885Ala Thr Asn Phe Ile Pro Pro Gln Glu Pro Se #r Ile Pro Asp Leu                890   #               895   #               900Gly Thr Asn Ile Ser Ala Ile Ser Leu Ala Il #e Phe Gly Leu Ala                905   #               910   #               915Val Ile Leu Ser Ile Phe Tyr Thr Arg Asn Ty #r Ile Arg Thr Gln                920   #               925   #               930Ile Gln Cys Tyr Thr Tyr Leu Val Asn Ile Ty #r Leu Lys Phe Asn                935   #               940   #               945Leu Leu Tyr Leu Leu Ser Ile Ile Lys Leu Il #e Ile Ile Tyr Lys                950   #               955   #               960Val Lys Tyr Lys Ser Cys Lys Phe Pro Asn Ty #r Leu Ile Asn Tyr                965   #               970   #               975Tyr Tyr Leu Ser Tyr Tyr Met Leu Ile Lys Me #t Ser Ile Ser Phe                980   #               985   #               990Pro Val Trp Asn Asn Pro Leu Ile Asn Phe                 995  #               1000 <210> SEQ ID NO 31 <211> LENGTH: 2970<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 31acctaaaacc ttgcaagttc aggaagaaac catctgcatc catattgaaa  #              50acctgacaca atgtatgcag caggctcagt gtgagtgaac tggaggcttc  #             100 tctacaac atg acc caa agg agc att gca ggt cct #att tgc aac             14 #4ctg aag ttt gtg act ctc ctg gtt gcc tta ag#t tca gaa ctc cca          18 #9ttc ctg gga gct gga gta cag ctt caa gac aa#t ggg tat aat gga          23 #4ttg ctc att gca att aat cct cag gta cct ga#g aat cag aac ctc          27 #9atc tca aac att aag gaa atg ata act gaa gc#t tca ttt tac cta          32 #4ttt aat gct acc aag aga aga gta ttt ttc ag#a aat ata aag att          36 #9tta ata cct gcc aca tgg aaa gct aat aat aa#c agc aaa ata aaa          41 #4caa gaa tca tat gaa aag gca aat gtc ata gt#g act gac tgg tat          45 #9ggg gca cat gga gat gat cca tac acc cta ca#a tac aga ggg tgt          50 #4gga aaa gag gga aaa tac att cat ttc aca cc#t aat ttc cta ctg          54 #9aat gat aac tta aca gct ggc tac gga tca cg#a ggc cga gtg ttt          59 #4gtc cat gaa tgg gcc cac ctc cgt tgg ggt gt#g ttc gat gag tat          63 #9aac aat gac aaa cct ttc tac ata aat ggg ca#a aat caa att aaa          68 #4gtg aca agg tgt tca tct gac atc aca ggc at#t ttt gtg tgt gaa          72 #9aaa ggt cct tgc ccc caa gaa aac tgt att at#t agt aag ctt ttt          77 #4aaa gaa gga tgc acc ttt atc tac aat agc ac#c caa aat gca act          81 #9gca tca ata atg ttc atg caa agt tta tct tc#t gtg gtt gaa ttt          86 #4tgt aat gca agt acc cac aac caa gaa gca cc#a aac cta cag aac          90 #9cag atg tgc agc ctc aga agt gca tgg gat gt#a atc aca gac tct          95 #4gct gac ttt cac cac agc ttt ccc atg aat gg#g act gag ctt cca          99 #9cct cct ccc aca ttc tcg ctt gta cag gct gg#t gac aaa gtg gtc         1044tgt tta gtg ctg gat gtg tcc agc aag atg gc#a gag gct gac aga         1089ctc ctt caa cta caa caa gcc gca gaa ttt ta#t ttg atg cag att         1134gtt gaa att cat acc ttc gtg ggc att gcc ag#t ttc gac agc aaa         1179gga gag atc aga gcc cag cta cac caa att aa#c agc aat gat gat         1224cga aag ttg ctg gtt tca tat ctg ccc acc ac#t gta tca gct aaa         1269aca gac atc agc att tgt tca ggg ctt aag aa#a gga ttt gag gtg         1314gtt gaa aaa ctg aat gga aaa gct tat ggc tc#t gtg atg ata tta         1359gtg acc agc gga gat gat aag ctt ctt ggc aa#t tgc tta ccc act         1404gtg ctc agc agt ggt tca aca att cac tcc at#t gcc ctg ggt tca         1449tct gca gcc cca aat ctg gag gaa tta tca cg#t ctt aca gga ggt         1494tta aag ttc ttt gtt cca gat ata tca aac tc#c aat agc atg att         1539gat gct ttc agt aga att tcc tct gga act gg#a gac att ttc cag         1584caa cat att cag ctt gaa agt aca ggt gaa aa#t gtc aaa cct cac         1629cat caa ttg aaa aac aca gtg act gtg gat aa#t act gtg ggc aac         1674gac act atg ttt cta gtt acg tgg cag gcc ag#t ggt cct cct gag         1719att ata tta ttt gat cct gat gga cga aaa ta#c tac aca aat aat         1764ttt atc acc aat cta act ttt cgg aca gct ag#t ctt tgg att cca         1809gga aca gct aag cct ggg cac tgg act tac ac#c ctg aac aat acc         1854cat cat tct ctg caa gcc ctg aaa gtg aca gt#g acc tct cgc gcc         1899tcc aac tca gct gtg ccc cca gcc act gtg ga#a gcc ttt gtg gaa         1944aga gac agc ctc cat ttt cct cat cct gtg at#g att tat gcc aat         1989gtg aaa cag gga ttt tat ccc att ctt aat gc#c act gtc act gcc         2034aca gtt gag cca gag act gga gat cct gtt ac#g ctg aga ctc ctt         2079gat gat gga gca ggt gct gat gtt ata aaa aa#t gat gga att tac         2124tcg agg tat ttt ttc tcc ttt gct gca aat gg#t aga tat agc ttg         2169aaa gtg cat gtc aat cac tct ccc agc ata ag#c acc cca gcc cac         2214tct att cca ggg agt cat gct atg tat gta cc#a ggt tac aca gca         2259aac ggt aat att cag atg aat gct cca agg aa#a tca gta ggc aga         2304aat gag gag gag cga aag tgg ggc ttt agc cg#a gtc agc tca gga         2349ggc tcc ttt tca gtg ctg gga gtt cca gct gg#c ccc cac cct gat         2394gtg ttt cca cca tgc aaa att att gac ctg ga#a gct gta aaa gta         2439gaa gag gaa ttg acc cta tct tgg aca gca cc#t gga gaa gac ttt         2484gat cag ggc cag gct aca agc tat gaa ata ag#a atg agt aaa agt         2529cta cag aat atc caa gat gac ttt aac aat gc#t att tta gta aat         2574aca tca aag cga aat cct cag caa gct ggc at#c agg gag ata ttt         2619acg ttc tca ccc cag att tcc acg aat gga cc#t gaa cat cag cca         2664aat gga gaa aca cat gaa agc cac aga att ta#t gtt gca ata cga         2709gca atg gat agg aac tcc tta cag tct gct gt#a tct aac att gcc         2754cag gcg cct ctg ttt att ccc ccc aat tct ga#t cct gta cct gcc         2799aga gat tat ctt ata ttg aaa gga gtt tta ac#a gca atg ggt ttg         2844ata gga atc att tgc ctt att ata gtt gtg ac#a cat cat act tta         2889agc agg aaa aag aga gca gac aag aaa gag aa#t gga aca aaa tta         2934tta taaataaata tccaaagtgt cttccttctc aaa      #                  #     2970 <210> SEQ ID NO 32 <211> LENGTH: 943 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 32Met Thr Gln Arg Ser Ile Ala Gly Pro Ile Cy #s Asn Leu Lys Phe1                5   #                 10  #                 15Val Thr Leu Leu Val Ala Leu Ser Ser Glu Le #u Pro Phe Leu Gly                 20  #                 25  #                 30Ala Gly Val Gln Leu Gln Asp Asn Gly Tyr As #n Gly Leu Leu Ile                 35  #                 40  #                 45Ala Ile Asn Pro Gln Val Pro Glu Asn Gln As #n Leu Ile Ser Asn                 50  #                 55  #                 60Ile Lys Glu Met Ile Thr Glu Ala Ser Phe Ty #r Leu Phe Asn Ala                 65  #                 70  #                 75Thr Lys Arg Arg Val Phe Phe Arg Asn Ile Ly #s Ile Leu Ile Pro                 80  #                 85  #                 90Ala Thr Trp Lys Ala Asn Asn Asn Ser Lys Il #e Lys Gln Glu Ser                 95  #                100  #                105Tyr Glu Lys Ala Asn Val Ile Val Thr Asp Tr #p Tyr Gly Ala His                110   #               115   #               120Gly Asp Asp Pro Tyr Thr Leu Gln Tyr Arg Gl #y Cys Gly Lys Glu                125   #               130   #               135Gly Lys Tyr Ile His Phe Thr Pro Asn Phe Le #u Leu Asn Asp Asn                140   #               145   #               150Leu Thr Ala Gly Tyr Gly Ser Arg Gly Arg Va #l Phe Val His Glu                155   #               160   #               165Trp Ala His Leu Arg Trp Gly Val Phe Asp Gl #u Tyr Asn Asn Asp                170   #               175   #               180Lys Pro Phe Tyr Ile Asn Gly Gln Asn Gln Il #e Lys Val Thr Arg                185   #               190   #               195Cys Ser Ser Asp Ile Thr Gly Ile Phe Val Cy #s Glu Lys Gly Pro                200   #               205   #               210Cys Pro Gln Glu Asn Cys Ile Ile Ser Lys Le #u Phe Lys Glu Gly                215   #               220   #               225Cys Thr Phe Ile Tyr Asn Ser Thr Gln Asn Al #a Thr Ala Ser Ile                230   #               235   #               240Met Phe Met Gln Ser Leu Ser Ser Val Val Gl #u Phe Cys Asn Ala                245   #               250   #               255Ser Thr His Asn Gln Glu Ala Pro Asn Leu Gl #n Asn Gln Met Cys                260   #               265   #               270Ser Leu Arg Ser Ala Trp Asp Val Ile Thr As #p Ser Ala Asp Phe                275   #               280   #               285His His Ser Phe Pro Met Asn Gly Thr Glu Le #u Pro Pro Pro Pro                290   #               295   #               300Thr Phe Ser Leu Val Gln Ala Gly Asp Lys Va #l Val Cys Leu Val                305   #               310   #               315Leu Asp Val Ser Ser Lys Met Ala Glu Ala As #p Arg Leu Leu Gln                320   #               325   #               330Leu Gln Gln Ala Ala Glu Phe Tyr Leu Met Gl #n Ile Val Glu Ile                335   #               340   #               345His Thr Phe Val Gly Ile Ala Ser Phe Asp Se #r Lys Gly Glu Ile                350   #               355   #               360Arg Ala Gln Leu His Gln Ile Asn Ser Asn As #p Asp Arg Lys Leu                365   #               370   #               375Leu Val Ser Tyr Leu Pro Thr Thr Val Ser Al #a Lys Thr Asp Ile                380   #               385   #               390Ser Ile Cys Ser Gly Leu Lys Lys Gly Phe Gl #u Val Val Glu Lys                395   #               400   #               405Leu Asn Gly Lys Ala Tyr Gly Ser Val Met Il #e Leu Val Thr Ser                410   #               415   #               420Gly Asp Asp Lys Leu Leu Gly Asn Cys Leu Pr #o Thr Val Leu Ser                425   #               430   #               435Ser Gly Ser Thr Ile His Ser Ile Ala Leu Gl #y Ser Ser Ala Ala                440   #               445   #               450Pro Asn Leu Glu Glu Leu Ser Arg Leu Thr Gl #y Gly Leu Lys Phe                455   #               460   #               465Phe Val Pro Asp Ile Ser Asn Ser Asn Ser Me #t Ile Asp Ala Phe                470   #               475   #               480Ser Arg Ile Ser Ser Gly Thr Gly Asp Ile Ph #e Gln Gln His Ile                485   #               490   #               495Gln Leu Glu Ser Thr Gly Glu Asn Val Lys Pr #o His His Gln Leu                500   #               505   #               510Lys Asn Thr Val Thr Val Asp Asn Thr Val Gl #y Asn Asp Ile Met                515   #               520   #               525Phe Leu Val Thr Trp Gln Ala Ser Gly Pro Pr #o Glu Ile Ile Leu                530   #               535   #               540Phe Asp Pro Asp Gly Arg Lys Tyr Tyr Thr As #n Asn Phe Thr Thr                545   #               550   #               555Asn Leu Thr Phe Arg Thr Ala Ser Leu Trp Il #e Pro Gly Thr Ala                560   #               565   #               570Lys Pro Gly His Trp Thr Tyr Thr Leu Asn As #n Thr His His Ser                575   #               580   #               585Leu Gln Ala Leu Lys Val Thr Val Thr Ser Ar #g Ala Ser Asn Ser                590   #               595   #               600Ala Val Pro Pro Ala Thr Val Glu Ala Phe Va #l Glu Arg Asp Ser                605   #               610   #               615Leu His Phe Pro His Pro Val Met Ile Tyr Al #a Asn Val Lys Gln                620   #               625   #               630Gly Phe Tyr Pro Ile Ile Asn Ala Thr Val Th #r Ala Thr Val Glu                635   #               640   #               645Pro Glu Thr Gly Asp Pro Val Thr Leu Arg Le #u Leu Asp Asp Gly                650   #               655   #               660Ala Gly Ala Asp Val Ile Lys Asn Asp Gly Il #e Tyr Ser Arg Tyr                665   #               670   #               675Phe Phe Ser Phe Ala Ala Asn Gly Arg Tyr Se #r Leu Lys Val His                680   #               685   #               690Val Asn His Ser Pro Ser Ile Ser Thr Pro Al #a His Ser Ile Pro                695   #               700   #               705Gly Ser His Ala Met Tyr Val Pro Gly Tyr Th #r Ala Asn Gly Asn                710   #               715   #               720Ile Gln Met Asn Ala Pro Arg Lys Ser Val Gl #y Arg Asn Glu Glu                725   #               730   #               735Glu Arg Lys Trp Gly Phe Ser Arg Val Ser Se #r Gly Gly Ser Phe                740   #               745   #               750Ser Val Leu Gly Val Pro Ala Gly Pro His Pr #o Asp Val Phe Pro                755   #               760   #               765Pro Cys Lys Ile Ile Asp Leu Glu Ala Val Ly #s Val Glu Glu Glu                770   #               775   #               780Leu Thr Leu Ser Trp Thr Ala Pro Gly Glu As #p Phe Asp Gln Gly                785   #               790   #               795Gln Ala Thr Ser Tyr Glu Ile Arg Met Ser Ly #s Ser Leu Gln Asn                800   #               805   #               810Ile Gln Asp Asp Phe Asn Asn Ala Ile Leu Va #l Asn Thr Ser Lys                815   #               820   #               825Arg Asn Pro Gln Gln Ala Gly Ile Arg Glu Il #e Phe Thr Phe Ser                830   #               835   #               840Pro Gln Ile Ser Thr Asn Gly Pro Glu His Gl #n Pro Asn Gly Glu                845   #               850   #               855Thr His Glu Ser His Arg Ile Tyr Val Ala Il #e Arg Ala Met Asp                860   #               865   #               870Arg Asn Ser Leu Gln Ser Ala Val Ser Asn Il #e Ala Gln Ala Pro                875   #               880   #               885Leu Phe Ile Pro Pro Asn Ser Asp Pro Val Pr #o Ala Arg Asp Tyr                890   #               895   #               900Leu Ile Leu Lys Gly Val Leu Thr Ala Met Gl #y Leu Ile Gly Ile                905   #               910   #               915Ile Cys Leu Ile Ile Val Val Thr His His Th #r Leu Ser Arg Lys                920   #               925   #               930Lys Arg Ala Asp Lys Lys Glu Asn Gly Thr Ly #s Leu Leu                935   #               940   #       943<210> SEQ ID NO 33 <211> LENGTH: 3022 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 33actggagcag tgcgacc atg gtg cca ggg ctg cag gtc #ctt ctg ttc            47 ctc acc ctg cat ctc ctg cag aac aca gag ag#c tcc atg gtg cat            #92ctc aac agc aat gga tac gag ggt gtg gtc at#t gcc att aac ccc          13 #7agt gtg cca gag gac gaa agg ctc atc cca ag#c ata aag gaa atg          18 #2gta act caa gct tct acc tac ctg ttt gaa gc#c agc caa gga aga          22 #7gtt tat ttc agg aac ata agc ata tta gtc cc#g atg acc tgg aag          27 #2tcg aaa tct gag tac tta atg cca aaa cga ga#a tcg tac gac aaa          31 #7gca gac gtc ata gtt gcg gat cct cac ctg ca#a cat gga gac gac          36 #2ccc tac acc ctt cag tat gga cag tgt ggg ga#c aga gga cag tac          40 #7ata cac ttc act cca aac ttc cta ctc act ga#t aac ttg cgt atc          45 #2tat gga ccc cga ggc aga gtc ttt gtc cat ga#g tgg gcc cat ctc          49 #7cgg tgg gga gta ttt gat gag tat aac gtg ga#c cgg tca ctt tac          54 #2att tct aga aag aac act ata gaa gca aca ag#g tgc tcc gcc agc          58 #7atc aca ggc aag aag gtg gtc cac gag tgt ca#g aga ggc agc tgt          63 #2gtg aca agg gcg tgc cgg cgt gac tcg aag ac#a cgg ctg tat gaa          67 #7ccc aaa tgt aca ttt atc cca gac aaa ata ca#g aca gct ggg gcc          72 #2tcc ata atg ttc atg caa aac ctc aat tct gt#g gtt gaa ttt tgc          76 #7aca gaa aat aac cac aat gca gaa gcc cca aa#c cta caa aac aaa          81 #2atg tgc aat cgc aga agc acg tgg gat gta at#c aag acg tct gct          85 #7gac ttt cag aat gcc cct ccc atg aga gga ac#a gaa gcc cct cct          90 #2cca cct aca ttt tat ctg ctc aag tcc aga ag#g cga gtg gtg tgc          94 #7ttg gtg ctg gat aaa tct gga agc atg gac aa#a gaa gac cgt ctt          99 #2att cga atg aat caa gca gca gaa ctg tac tt#a act caa att gtg         1037gaa aag gag tct atg gtt gga tta gtc aca tt#t gac agc gct gcc         1082cac atc caa aat tat cta ata aaa ata acg ag#t agt agt gac tac         1127caa aag atc acc gca aac ctc ccc caa cag gc#t tct ggt gga act         1172tca att tgc cat gga ctc cag gca gga ttt ca#g gca att acc tcc         1217agt gac cag agc act tcc ggt tct gag atc gt#a ttg ctg aca gat         1262ggg gaa gat aat gga ata cgt tcc tgc ttt ga#g gcc gtc tct cgc         1307agc ggt gcc atc atc cac acc atc gct ctg gg#g cct tcg cgt gcc         1352cga gaa ctg gag act ctg tcg gac atg aca gg#a ggg ctt cgt ttc         1397tat gcc aac aaa gac cta aac agc ctt atc ga#t gct ttc agt aga         1442att tca tct aca agt ggc agc gtc tcc cag ca#g gct ctg cag ttg         1487gag agc aaa gcc ttc gat gtc aga gca ggg gc#a tgg ata aac ggt         1532aca gta cct ctg gac agt acc gtc ggc aac ga#c acg ttc ttt gtt         1577atc acc tgg atg gta aaa aag cca gaa atc at#t ctt caa gat cca         1622aaa gga aaa aaa tat aca acc tca gat ttc ca#a gat gat aaa cta         1667aac atc cgg tct gct aga ctt caa ata ccg gg#c act gca gag aca         1712ggt act tgg act tac agc tac acg ggt acc aa#g tct cag ttg att         1757aca atg aca gtg acc act cga gca aga agt cc#c acc atg gaa cca         1802ctc ctg ggc tac tgc tac atg agt cag agc ac#a gcc cag tac cct         1847agc cgg atg att gtg tac gca cgg gtc agc ca#a gga ttt ttg cct         1892gtt ctg gga gcc aat gtc aca gcc ctc ata ga#a gct gaa cat gga         1937cat caa gtc acc ttg gag ctc tgg gac aat gg#g gca ggt gct gat         1982atc gtt aaa aat gat ggc atc tac aca aga ta#c ttt aca gat tat         2027cat gga aat ggt aga tac agc cta aaa gtg cg#t gtc cag gca caa         2072aga aac aaa acc aga ctg agc tta aga cag aa#g aac aag tct tta         2117tat ata cct ggc tat gtg gaa aat ggt aaa at#t gta ctg aat cca         2162ccc aga cca gat gtc caa gaa gaa gcc ata ga#a gct aca gtg gaa         2207gac ttc aac aga gta acc tct gga ggg tcg tt#t act gtg tct gga         2252gcg ccc cct gat ggc gac cac gct cgt gtg tt#c cca cca agt aaa         2297gtc aca gac ctg gag gct gag ttt ata ggt ga#t tat att cac ctt         2342aca tgg acg gcc cct ggc aag gtt ctc gac aa#t gga aga gca cat         2387aga tac atc atc aga atg agc cag cat cct ct#g gat ctc caa gaa         2432gat ttt aac aat gct act tta gtg aat gct tc#c agt ctg ata cct         2477aaa gaa gct ggc tca aaa gaa gca ttt aaa tt#c aaa cca gaa act         2522ttt aaa ata gca aat ggc atc cag ctc tac at#t gca atc cag gca         2567gac aat gaa gcc agt ctc acc tct gag gtc tc#c aac atc gca cag         2612gct gtc aag ctt act tct cta gaa gat agt at#c tct gca ctg ggt         2657gat gat att tct gca atc tct atg aca att tg#g ggg tta act gtg         2702att ttt aac tct att tta aac tagaagatag aatggcact #a                  # 2743 aaatgcaatc ctgtacatat ttgctaagtg ttgctttaga atgtctttac  #            2793tacacactca aaggctgcct gtcaacaatt gtaatataga agttcatatt  #            2843caaagttgaa aatcccgagt tactaacaca attcttttgc tatatgtaga  #            2893tcaagattaa cagttcctca ttcaatttct taattgttcc atttactatg  #            2943gaaataagat atccattctc ttttcacagt gtgatgcaag ttcactttgt  #            2993 atatgaaaat aaaaaatttg tacaactcg         #                   #          3022 <210> SEQ ID NO 34 <211> LENGTH: 902<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 34Met Val Pro Gly Leu Gln Val Leu Leu Phe Le #u Thr Leu His Leu                 5  #                   #10                   #15Leu Gln Asn Thr Glu Ser Ser Met Val His Le #u Asn Ser Asn Gly                 20  #                 25  #                 30Tyr Glu Gly Val Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp                 35  #                 40  #                 45Glu Arg Leu Ile Pro Ser Ile Lys Glu Met Va #l Thr Gln Ala Ser                 50  #                 55  #                 60Thr Tyr Leu Phe Glu Ala Ser Gln Gly Arg Va #l Tyr Phe Arg Asn                 65  #                 70  #                 75Ile Ser Ile Leu Val Pro Met Thr Trp Lys Se #r Lys Ser Glu Tyr                 80  #                 85  #                 90Leu Met Pro Lys Arg Glu Ser Tyr Asp Lys Al #a Asp Val Ile Val                 95  #                100  #                105Ala Asp Pro His Leu Gln His Gly Asp Asp Pr #o Tyr Thr Leu Gln                110   #               115   #               120Tyr Gly Gln Cys Gly Asp Arg Gly Gln Tyr Il #e His Phe Thr Pro                125   #               130   #               135Asn Phe Leu Leu Thr Asp Asn Leu Arg Ile Ty #r Gly Pro Arg Gly                140   #               145   #               150Arg Val Phe Val His Glu Trp Ala His Leu Ar #g Trp Gly Val Phe                155   #               160   #               165Asp Glu Tyr Asn Val Asp Arg Ser Pro Tyr Il #e Ser Arg Lys Asn                170   #               175   #               180Thr Ile Glu Ala Thr Arg Cys Ser Ala Ser Il #e Thr Gly Lys Lys                185   #               190   #               195Val Val His Glu Cys Gln Arg Gly Ser Cys Va #l Thr Arg Ala Cys                200   #               205   #               210Arg Arg Asp Ser Lys Thr Arg Leu Tyr Glu Pr #o Lys Cys Thr Phe                215   #               220   #               225Ile Pro Asp Lys Ile Gln Thr Ala Gly Ala Se #r Ile Met Phe Met                230   #               235   #               240Gln Asn Leu Asn Ser Val Val Glu Phe Cys Th #r Glu Asn Asn His                245   #               250   #               255Asn Ala Glu Ala Pro Asn Leu Gln Asn Lys Me #t Cys Asn Arg Arg                260   #               265   #               270Ser Thr Trp Asp Val Ile Lys Thr Ser Ala As #p Phe Gln Asn Ala                275   #               280   #               285Pro Pro Met Arg Gly Thr Glu Ala Pro Pro Pr #o Pro Thr Phe Tyr                290   #               295   #               300Leu Leu Lys Ser Arg Arg Arg Val Val Cys Le #u Val Leu Asp Lys                305   #               310   #               315Ser Gly Ser Met Asp Lys Glu Asp Arg Leu Il #e Arg Met Asn Gln                320   #               325   #               330Ala Ala Glu Leu Tyr Leu Thr Gln Ile Val Gl #u Lys Glu Ser Met                335   #               340   #               345Val Gly Leu Val Thr Phe Asp Ser Ala Ala Hi #s Ile Gln Asn Tyr                350   #               355   #               360Leu Ile Lys Ile Thr Ser Ser Ser Asp Tyr Gl #n Lys Ile Thr Ala                365   #               370   #               375Asn Leu Pro Gln Gln Ala Ser Gly Gly Thr Se #r Ile Cys His Gly                380   #               385   #               390Leu Gln Ala Gly Phe Gln Ala Ile Thr Ser Se #r Asp Gln Ser Thr                395   #               400   #               405Ser Gly Ser Glu Ile Val Leu Leu Thr Asp Gl #y Glu Asp Asn Gly                410   #               415   #               420Ile Arg Ser Cys Phe Glu Ala Val Ser Arg Se #r Gly Ala Ile Ile                425   #               430   #               435His Thr Ile Ala Leu Gly Pro Ser Arg Ala Ar #g Glu Leu Glu Thr                440   #               445   #               450Leu Ser Asp Met Thr Gly Gly Leu Arg Phe Ty #r Ala Asn Lys Asp                455   #               460   #               465Leu Asn Ser Leu Ile Asp Ala Phe Ser Arg Il #e Ser Ser Thr Ser                470   #               475   #               480Gly Ser Val Ser Gln Gln Ala Leu Gln Leu Gl #u Ser Lys Ala Phe                485   #               490   #               495Asp Val Arg Ala Gly Ala Trp Ile Asn Gly Th #r Val Pro Leu Asp                500   #               505   #               510Ser Thr Val Gly Asn Asp Thr Phe Phe Val Il #e Thr Trp Met Val                515   #               520   #               525Lys Lys Pro Glu Ile Ile Leu Gln Asp Pro Ly #s Gly Lys Lys Tyr                530   #               535   #               540Thr Thr Ser Asp Phe Gln Asp Asp Lys Leu As #n Ile Arg Ser Ala                545   #               550   #               555Arg Leu Gln Ile Pro Gly Thr Ala Glu Thr Gl #y Thr Trp Thr Tyr                560   #               565   #               570Ser Tyr Thr Gly Thr Lys Ser Gln Leu Ile Th #r Met Thr Val Thr                575   #               580   #               585Thr Arg Ala Arg Ser Pro Thr Met Glu Pro Le #u Leu Gly Tyr Cys                590   #               595   #               600Tyr Met Ser Gln Ser Thr Ala Gln Tyr Pro Se #r Arg Met Ile Val                605   #               610   #               615Tyr Ala Arg Val Ser Gln Gly Phe Leu Pro Va #l Leu Gly Ala Asn                620   #               625   #               630Val Thr Ala Leu Ile Glu Ala Glu His Gly Hi #s Gln Val Thr Leu                635   #               640   #               645Glu Leu Trp Asp Asn Gly Ala Gly Ala Asp Il #e Val Lys Asn Asp                650   #               655   #               660Gly Ile Tyr Thr Arg Tyr Phe Thr Asp Tyr Hi #s Gly Asn Gly Arg                665   #               670   #               675Tyr Ser Leu Lys Val Arg Val Gln Ala Gln Ar #g Asn Lys Thr Arg                680   #               685   #               690Leu Ser Leu Arg Gln Lys Asn Lys Ser Leu Ty #r Ile Pro Gly Tyr                695   #               700   #               705Val Glu Asn Gly Lys Ile Val Leu Asn Pro Pr #o Arg Pro Asp Val                710   #               715   #               720Gln Glu Glu Ala Ile Glu Ala Thr Val Glu As #p Phe Asn Arg Val                725   #               730   #               735Thr Ser Gly Gly Ser Phe Thr Val Ser Gly Al #a Pro Pro Asp Gly                740   #               745   #               750Asp His Ala Arg Val Phe Pro Pro Ser Lys Va #l Thr Asp Leu Glu                755   #               760   #               765Ala Glu Phe Ile Gly Asp Tyr Ile His Leu Th #r Trp Thr Ala Pro                770   #               775   #               780Gly Lys Val Leu Asp Asn Gly Arg Ala His Ar #g Tyr Ile Ile Arg                785   #               790   #               795Met Ser Gln His Pro Leu Asp Leu Gln Glu As #p Phe Asn Asn Ala                800   #               805   #               810Thr Leu Val Asn Ala Ser Ser Leu Ile Pro Ly #s Glu Ala Gly Ser                815   #               820   #               825Lys Glu Ala Phe Lys Phe Lys Pro Glu Thr Ph #e Lys Ile Ala Asn                830   #               835   #               840Gly Ile Gln Leu Tyr Ile Ala Ile Gln Ala As #p Asn Glu Ala Ser                845   #               850   #               855Leu Thr Ser Glu Val Ser Asn Ile Ala Gln Al #a Val Lys Leu Thr                860   #               865   #               870Ser Leu Glu Asp Ser Ile Ser Ala Leu Gly As #p Asp Ile Ser Ala                875   #               880   #               885Ile Ser Met Thr Ile Trp Gly Leu Thr Val Il #e Phe Asn Ser Ile                890   #               895   #               900 Leu Asn    902 <210> SEQ ID NO 35 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 35gaaccttgcc aggggccg              #                   #                  #  18 <210> SEQ ID NO 36 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 36ccacgtgctt ctgcgattgc ac            #                  #                 22 <210> SEQ ID NO 37 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 37gcggccgcaa tggggccatt taagagttct g         #                  #          31 <210> SEQ ID NO 38 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 38gcggccgcag ccctaggcta ttgacagctg          #                  #           30 <210> SEQ ID NO 39 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 39agaatcaaga tgaacacaga actc           #                  #                24 <210> SEQ ID NO 40 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 40caaggtattt cacaacttat gacacg           #                  #              26 <210> SEQ ID NO 41 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 41gcggccgcta caacatgacc caaaggagc          #                  #            29 <210> SEQ ID NO 42 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 42gcggccgcga cactttggat atttatttat aataattttg ttc     #                  # 43 <210> SEQ ID NO 43 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 43cctttatgtt ttgaatgag              #                  #                   # 19 <210> SEQ ID NO 44 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 44caactatgac atctgcctgg tc            #                  #                 22 <210> SEQ ID NO 45 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Amplification primer <400> SEQUENCE: 45cacaaagcta ggctaagtca agaac           #                  #               25 <210> SEQ ID NO 46 <211> LENGTH: 903 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Calcium sensitive chloride #channel from bovine       tracheal epithelium (Cunningham et a#l., 1995, J. Biol Chem <400> SEQUENCE: 46Met Val Pro Arg Leu Thr Val Ile Leu Phe Le #u Thr Leu His Leu                 5  #                   #10                   #15Leu Pro Gly Met Lys Ser Ser Met Val Asn Le #u Ile Asn Asn Gly                 20  #                 25  #                 30Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Se #r Val Pro Glu Asp                 35  #                 40  #                 45Glu Lys Leu Ile Gln Asn Ile Lys Glu Met Va #l Thr Glu Ala Ser                 50  #                 55  #                 60Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Va #l Tyr Phe Arg Asn                 65  #                 70  #                 75Val Ser Ile Leu Ile Pro Met Thr Trp Lys Se #r Lys Ser Glu Tyr                 80  #                 85  #                 90Leu Met Pro Lys Gln Glu Ser Tyr Asp Gln Al #a Glu Val Ile Val                 95  #                100  #                105Ala Asn Pro Tyr Leu Lys His Gly Asp Asp Pr #o Tyr Thr Leu Gln                110   #               115   #               120Tyr Gly Arg Cys Gly Glu Lys Gly Gln Tyr Il #e His Phe Thr Pro                125   #               130   #               135Asn Phe Leu Leu Thr Asn Asn Leu Pro Ile Ty #r Gly Ser Arg Gly                140   #               145   #               150Arg Ala Phe Val His Glu Trp Ala His Leu Ar #g Trp Gly Ile Phe                155   #               160   #               165Asp Glu Tyr Asn Gly Asp Gln Pro Phe Tyr Il #e Ser Arg Arg Asn                170   #               175   #               180Thr Ile Glu Ala Thr Arg Cys Ser Thr His Il #e Thr Gly Thr Asn                185   #               190   #               195Val Ile Val Lys Cys Gln Gly Gly Ser Cys Il #e Thr Arg Pro Cys                200   #               205   #               210Arg Arg Asp Ser Gln Thr Gly Leu Tyr Glu Al #a Lys Cys Thr Phe                215   #               220   #               225Ile Pro Glu Lys Ser Gln Thr Ala Arg Glu Se #r Ile Met Phe Met                230   #               235   #               240Gln Ser Leu His Ser Val Thr Glu Phe Cys Th #r Glu Lys Thr His                245   #               250   #               255Asn Val Glu Ala Pro Asn Leu Gln Asn Lys Me #t Cys Asn Gly Lys                260   #               265   #               270Ser Thr Trp Asp Val Ile Met Asn Ser Thr As #p Phe Gln Asn Thr                275   #               280   #               285Ser Pro Met Thr Glu Met Asn Pro Pro Thr Gl #n Pro Thr Phe Ser                290   #               295   #               300Leu Leu Lys Ser Lys Gln Arg Val Val Cys Le #u Val Leu Asp Lys                305   #               310   #               315Ser Gly Ser Met Ser Ser Glu Asp Arg Leu Ph #e Arg Met Asn Gln                320   #               325   #               330Ala Ala Glu Leu Phe Leu Ile Gln Ile Ile Gl #u Lys Gly Ser Leu                335   #               340   #               345Val Gly Met Val Thr Phe Asp Ser Val Ala Gl #u Ile Arg Asn Asn                350   #               355   #               360Leu Thr Lys Ile Thr Asp Asp Asn Val Tyr Gl #u Asn Ile Thr Ala                365   #               370   #               375Asn Leu Pro Gln Glu Ala Asn Gly Gly Thr Se #r Ile Cys Arg Gly                380   #               385   #               390Leu Lys Ala Gly Phe Gln Ala Ile Ile Gln Se #r Gln Gln Ser Thr                395   #               400   #               405Ser Gly Ser Glu Ile Ile Leu Leu Thr Asp Gl #y Glu Asp Asn Glu                410   #               415   #               420Ile His Ser Cys Ile Glu Glu Val Lys Gln Se #r Gly Val Ile Ile                425   #               430   #               435His Thr Val Ala Leu Gly Pro Ser Ala Ala Ly #s Glu Leu Glu Thr                440   #               445   #               450Leu Ser Asp Met Thr Gly Gly His Arg Phe Ty #r Ala Asn Lys Asp                455   #               460   #               465Ile Asn Gly Leu Thr Asn Ala Phe Ser Arg Il #e Ser Ser Arg Ser                470   #               475   #               480Gly Ser Ile Thr Gln Gln Thr Ile Gln Leu Gl #u Ser Lys Ala Leu                485   #               490   #               495Ala Ile Thr Glu Lys Lys Trp Val Asn Gly Th #r Val Pro Val Asp                500   #               505   #               510Ser Thr Ile Gly Asn Asp Thr Phe Phe Val Va #l Thr Trp Thr Ile                515   #               520   #               525Lys Lys Pro Glu Ile Leu Leu Gln Asp Pro Ly #s Gly Lys Lys Tyr                530   #               535   #               540Lys Thr Ser Asp Phe Lys Glu Asp Lys Leu As #n Ile His Ser Ala                545   #               550   #               555Arg Leu Arg Ile Pro Gly Ile Ala Glu Thr Gl #y Thr Trp Thr Tyr                560   #               565   #               570Ser Leu Leu Asn Asn His Ala Ser Pro Gln Il #e Leu Thr Val Thr                575   #               580   #               585Val Thr Thr Arg Ala Arg Ser Pro Thr Thr Pr #o Pro Val Thr Ala                590   #               595   #               600Thr Ala His Met Asn Gln Asn Thr Ala His Ty #r Pro Ser Pro Val                605   #               610   #               615Ile Val Tyr Ala Gln Val Ser Gln Gly Phe Le #u Pro Val Leu Gly                620   #               625   #               630Ile Asn Val Thr Ala Ile Ile Glu Thr Glu As #p Gly His Gln Val                635   #               640   #               645Thr Leu Glu Leu Trp Asp Asn Gly Ala Gly Al #a Asp Ala Thr Lys                650   #               655   #               660Asp Asp Gly Val Tyr Ser Arg Tyr Phe Thr Th #r Tyr Asp Thr Asn                665   #               670   #               675Gly Arg Tyr Ser Val Lys Val His Ala Glu Al #a Arg Asn Asn Thr                680   #               685   #               690Ala Arg Leu Ser Leu Arg Gln Pro Gln Asn Ly #s Ala Leu Tyr Ile                695   #               700   #               705Pro Gly Tyr Ile Glu Asn Gly Lys Ile Ile Le #u Asn Pro Pro Arg                710   #               715   #               720Pro Glu Val Lys Asp Asp Leu Ala Lys Ala Gl #u Ile Glu Asp Phe                725   #               730   #               735Ser Arg Leu Thr Ser Gly Gly Ser Phe Thr Va #l Ser Gly Ala Pro                740   #               745   #               750Pro Gly Asn His Pro Ser Val Leu Pro Pro As #n Lys Ile Thr Asp                755   #               760   #               765Leu Glu Ala Lys Phe Lys Glu Asp His Ile Gl #n Leu Ser Trp Thr                770   #               775   #               780Ala Pro Ala Asn Val Leu Asp Lys Gly Lys Al #a Asn Ser Tyr Ile                785   #               790   #               795Ile Arg Ile Ser Lys Ser Phe Leu Asp Leu Gl #n Lys Asp Phe Asp                800   #               805   #               810Asn Ala Thr Leu Val Asn Thr Ser Ser Leu Ly #s Pro Lys Glu Ala                815   #               820   #               825Gly Ser Asp Glu Asn Phe Glu Phe Lys Pro Gl #u Pro Phe Arg Ile                830   #               835   #               840Glu Asn Gly Thr Asn Phe Tyr Ile Ala Val Gl #n Ala Ile Asn Glu                845   #               850   #               855Ala Asn Leu Thr Ser Glu Val Ser Asn Ile Al #a Gln Ala Ile Lys                860   #               865   #               870Phe Ile Pro Met Pro Glu Asp Ser Val Pro Al #a Leu Gly Thr Lys                875   #               880   #               885Ile Ser Ala Ile Asn Leu Ala Ile Phe Ala Le #u Ala Met Ile Leu                890   #               895   #               900Ser Ile Val         903 <210> SEQ ID NO 47 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<223> OTHER INFORMATION: partial sequence of human # c-myc protein<400> SEQUENCE: 47 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu1               5    #                 10

We claim:
 1. An isolated nucleic acid molecule comprising a nucleotidesequence encoding a protein having an amino acid sequence of SEQ ID NO:28.
 2. The nucleic acid molecule of claim 1 having a sequence of SEQ IDNO:
 27. 3. A vector comprising the nucleic acid molecule of claim
 1. 4.A recombinant vector containing the nucleic acid molecule according toclaim 1 wherein the nucleic acid molecule is operably linked to one ormore control elements.
 5. A host cell containing the vector of claim 4.6. A method of providing a calcium activated channel chloride activityto a mammalian cell comprising transfecting the mammalian cell with thevector of claim
 4. 7. An isolated and purified polypeptide comprisingthe amino acid sequence of SEQ ID NO: 28.